α-Bungarotoxin labelling of AMPA receptor-associated TARPs in living neurons.

In mammalian central neurons AMPARs are clustered at glutamatergic synapses where they mediate fast excitatory transmission. In addition to four pore-forming subunits (GluA1-4), AMPARs contain auxiliary transmembrane AMPAR regulatory proteins (γ2, γ3, γ4, γ5, γ7 or γ8) whose incorporation can vary between neuron types, brain regions, and stages of development. As well as modulating the functional properties of AMPARs, these auxiliary subunits play a central role in AMPAR trafficking. Directly visualizing TARPs could therefore provide a valuable insight into mechanisms underlying these processes. Although antibodies are routinely used for the detection of surface proteins, our experience suggests anti-TARP antibodies are too bulky to access their target, possibly due to close interactions between the extracellular domains of TARP and AMPAR subunits. We therefore assessed the utility of a small monovalent probe - fluorescent α-bungarotoxin (α-Btx) - for TARP labelling in living neurons. We inserted the bungarotoxin binding site (BBS) within the extracellular domain of TARPs to enable their detection in cells exposed to fluorescent α-Btx. Focusing on the prototypical TARP γ2, we demonstrate that the small size of fluorescent α-Btx allows it to bind to the BBS-tagged TARP when associated with AMPARs. Importantly, labelled γ2 enhances AMPAR function in the same way as unmodified γ2. In living neurons, fluorescent α-Btx-labelled γ2 associates with AMPAR clusters at synapses. As a proof-of-principle, we employed our method to compare the surface trafficking of γ2 and γ7 in cerebellar stellate neurons. Our approach provides a simple way to visualize TARPs within AMPARs in living cells.

A genetically engineered neuronal membrane-based nanotoxoid elicits protective immunity against neurotoxins.

Given their dangerous effects on the nervous system, neurotoxins represent a significant threat to public health. Various therapeutic approaches, including chelating agents, receptor decoys, and toxin-neutralizing antibodies, have been explored. While prophylactic vaccines are desirable, it is oftentimes difficult to effectively balance their safety and efficacy given the highly dangerous nature of neurotoxins. To address this, we report here on a nanovaccine against neurotoxins that leverages the detoxifying properties of cell membrane-coated nanoparticles. A genetically modified cell line with constitutive overexpression of the α7 nicotinic acetylcholine receptor is developed as a membrane source to generate biomimetic nanoparticles that can effectively and irreversibly bind to α-bungarotoxin, a model neurotoxin. This abrogates the biological activity of the toxin, enabling the resulting nanotoxoid to be safely delivered into the body and processed by the immune system. When co-administered with an immunological adjuvant, a strong humoral response against α-bungarotoxin is generated that protects vaccinated mice against a lethal dose of the toxin. Overall, this work highlights the potential of using genetic modification strategies to develop nanotoxoid formulations against various biological threats.

Development and optimization of a DNA aptamer to delay ß-bungarotoxin-induced lethality in a rodent model.

Current treatment of snakebite relies on immunoglobulin-rich antivenoms. However, production of these antivenoms is complicated and costly. Aptamers - single-stranded DNAs or RNAs with specific folding structures that bind to specific target molecules - represent excellent alternatives or complements to antibody-based therapeutics. However, no studies have systematically assessed the feasibility of using aptamers to mitigate venom-induced toxicity in vivo. ß-bungarotoxin is the predominant protein responsible for the toxicity of the venom of Bungarus multicinctus, a prominent venomous snake inhabiting Taiwan. In this study, we reported the screening and optimization of a DNA aptamer against ß-bungarotoxin and tested its utility in a mouse model. After 14 rounds of directed evolution of ligands by exponential enrichment, an aptamer, called BB3, displaying remarkable binding affinity and specificity for ß-bungarotoxin was obtained. Following structural prediction and point-modification experiments, BB3 underwent truncation and was modified with 2'-O-methylation and a 3'-inverted dT. This optimized aptamer showed sustained, high-affinity binding for ß-bungarotoxin and exhibited remarkable nuclease resistance in plasma. Importantly, administration of this optimized aptamer extended the survival time of mice treated with a lethal dose of ß-bungarotoxin. Collectively, our data provide a compelling illustration of the potential of aptamers as promising candidates for development of recombinant antivenom therapies.

Protein Painting Mass Spectrometry in the Discovery of Interaction Sites within the Acetylcholine Binding Protein.

Nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channel receptors that contribute to cognition, memory, and motor control in many organisms. The pharmacological targeting of these receptors, using small molecules or peptides, presents an important strategy for the development of drugs that can treat important human diseases, including neurodegenerative disorders. The Aplysia californica acetylcholine binding protein (Ac-AChBP) is a structural surrogate of the nAChR with high homology to the extracellular ligand binding domain of homopentameric nAChRs. In this study, we optimized protein-painting-based mass spectrometry to identify regions of interaction between the Ac-AChBP and several nAChR ligands. Using molecular dyes that adhere to the surface of a solubilized Ac-AChBP complex, we identified amino acid residues that constitute a contact site within the Ac-AChBP for α-bungarotoxin, choline, nicotine, and amyloid-ß 1-42. By integrating innovation in protein painting mass spectrometry with computational structural modeling, we present a new experimental tool for analyzing protein interactions of the nAChR.

Development of antibody-detection ELISA based on beta-bungarotoxin for evaluation of the neutralization potency of equine plasma against Bungarus multicinctus in Taiwan.

Animal testing has been the primary approach to assess the neutralization potency of antivenom for decades. However, the necessity to sacrifice large numbers of experimental animals during this process has recently raised substantial welfare concerns. Furthermore, the laborious and expensive nature of animal testing highlights the critical need to develop alternative in vitro assays. Here, we developed an antibody-detection enzyme-linked immunosorbent assay (ELISA) technique as an alternative approach to evaluate the neutralization potency of hyperimmunized equine plasma against B. multicinctus, a medically important venomous snake in Taiwan. Firstly, five major protein components of B. multicinctus venom, specifically, α-BTX, ß-BTX, γ-BTX, MTX, and NTL, were isolated. To rank their relative medical significance, a toxicity score system was utilized. Among the proteins tested, ß-BTX presenting the highest score was regarded as the major toxic component. Subsequently, antibody-detection ELISA was established based on the five major proteins and used to evaluate 55 hyperimmunized equine plasma samples with known neutralization potency. ELISA based on ß-BTX, the most lethal protein according to the toxicity score, exhibited the best sensitivity (75.6 %) and specificity (100 %) in discriminating between high-potency and low-potency plasma, supporting the hypothesis that highly toxic proteins offer better discriminatory power for potency evaluation. Additionally, a phospholipase A2 (PLA2) competition process was implemented to eliminate the antibodies targeting toxicologically irrelevant domains. This optimization greatly enhanced the performance of our assay, resulting in sensitivity of 97.6 % and specificity of 92.9 %. The newly developed antibody-detection ELISA presents a promising alternative to in vivo assays to determine the neutralization potency of antisera against B. multicinctus during the process of antivenom production.

Synthesis and Evaluation of Compound Targeting α7 and ß2 Subunits in Nicotinic Acetylcholinergic Receptor.

Nicotinic acetylcholine receptors (nAChRs) are involved in various central nervous system functions and have also been implicated in several neurodegenerative disorders. The heteromeric α4ß2* and homomeric α7 are two major nAChR subtypes which have been studied in the brain using positron emission tomography (PET). Our comparative autoradiographic studies of the two receptor types in the mouse and rat brains show major differences in the thalamus (α4ß2* >> α7), hippocampus (α7 >> α4ß2*), and subiculum (α4ß2* >> α7). A relatively newer heteromeric α7ß2 nAChR subtype has been identified in the brain which may have a greater role in neurodegeneration. We report the development of KS7 (3-(2-(S)-azetidinylmethoxy)-5-(1,4-diaza-bicyclo[3.2.2]nonane)pyridine) which incorporates structural features of Nifzetidine (high affinity for α4ß2* nAChR) and ASEM (high affinity for α7 nAChR) in an effort to target α7 and ß2 subunits in α7ß2 nAChR. KS7 exhibited higher affinities (IC50 = 50 to 172 nM) for [3H]cytisine radiolabeled sites and weaker affinities (IC50 = 10 µM) for [125I]-α-bungarotoxin radiolabeled rat brain sites in several brain regions. The weaker affinity of KS7 to α7 nAChR may suggest lack of binding at the α7 subunit of α7ß2 nAChR. A radiolabeled derivative of KS7 may be required to identify any specific binding to brain regions suggested to contain α7ß2 nAChR.

γ-Bungarotoxin impairs the vascular endothelial barrier function by inhibiting integrin α5.

γ-bungarotoxin (γ-BGT) is an RGD motif-containing protein, derived from the venom of Bungarus multicinctus, leading to acute death in mice. These RGD motif-containing proteins from snake venom belonging to the disintegrin family can interfere with vascular endothelial homeostasis by directly binding cell surface integrins. Targeting integrins that generate vascular endothelial dysfunction may contribute to γ-BGT poisoning, however, the underlying mechanisms have not been investigated in detail. In this study, the results showed that γ-BGT played a role in -promoting the permeability of the vascular endothelial barrier. Depending on its selective binding to integrin α5 in vascular endothelium (VE), γ-BGT initiated downstream events, including focal adhesion kinase dephosphorylation and cytoskeleton remodeling, resulting in the intercellular junction interruption. Those alternations facilitated paracellular permeability of VE and barrier dysfunction. Proteomics profiling identified that as a downstream effector of the integrin α5 / FAK signaling pathway cyclin D1 partially mediated the cellular structural changes and barrier dysfunction. Furthermore, VE-released plasminogen activator urokinase and platelet-derived growth factor D could serve as potential diagnostic biomarkers for γ-BGT-induced vascular endothelial dysfunction. Our results indicate the mechanisms through which γ-BGT as a novel disintegrin directly interacts with the VE, with consequences for barrier dysfunction.

The Potassium Channel Blocker ß-Bungarotoxin from the Krait Bungarus multicinctus Venom Manifests Antiprotozoal Activity.

Protozoal infections are a world-wide problem. The toxicity and somewhat low effectiveness of the existing drugs require the search for new ways of protozoa suppression. Snake venom contains structurally diverse components manifesting antiprotozoal activity; for example, those in cobra venom are cytotoxins. In this work, we aimed to characterize a novel antiprotozoal component(s) in the Bungarus multicinctus krait venom using the ciliate Tetrahymena pyriformis as a model organism. To determine the toxicity of the substances under study, surviving ciliates were registered automatically by an original BioLaT-3.2 instrument. The krait venom was separated by three-step liquid chromatography and the toxicity of the obtained fractions against T. pyriformis was analyzed. As a result, 21 kDa protein toxic to Tetrahymena was isolated and its amino acid sequence was determined by MALDI TOF MS and high-resolution mass spectrometry. It was found that antiprotozoal activity was manifested by ß-bungarotoxin (ß-Bgt) differing from the known toxins by two amino acid residues. Inactivation of ß-Bgt phospholipolytic activity with p-bromophenacyl bromide did not change its antiprotozoal activity. Thus, this is the first demonstration of the antiprotozoal activity of ß-Bgt, which is shown to be independent of its phospholipolytic activity.

Neurophysiological action of centrally-acting spider toxin polypeptides derived from Hadronyche versuta and Tegenaria agrestis venoms.

Established dogma concerning the action of insecticidal arthropod-derived peptides (e.g., scorpion toxins), was that they acted on the peripheral nervous system and were excluded from the central nervous system (CNS) by barrier systems. Initial evidence for a CNS-directed toxicological effect following parenteral administration was for a novel peptide from the Hobo spider, Tegeneria agrestis. This toxin was inactive on peripheral sensory and motor nerves, but had a potent excitatory effect on the CNS of larval Musca domestica. Recently, a commercialized formulation of GS-omega/kappa-Hxtx-Hv1a (HXTX), derived from the venom of the Australian blue mountain funnel web spider (Hadronyche versuta) was introduced for use in agriculture by Vestaron Corp. Its primary mode of action was found to be central neuroexcitation via positive allosteric modulation of nicotinic acetylcholine receptors (nAchR) of cockroach neurons. In the present study, this peptide showed hyperexcitation followed by a decrease in firing of the Drosophila melanogaster larval CNS that was prevented by co-exposure to 100 nM α-bungarotoxin (α-BGTX), a classical nAchR noncompetitive antagonist. This effect was mirrored in isobologram analysis, which showed clear antagonism between the two toxins when injected into adult houseflies. Interestingly, U1-agatoxin-Ta1b-QA derived from Tegeneria agrestis (VST-7304) had a similar biphasic action, but showed increased nerve discharge when co-exposed with 100 nM α-BGTX, and had additive effects when injected together with α-BGTX in isobologram analyses. Binary mixtures of HXTX or VST-7304 with 30 nM nicotine showed clear evidence of synergized nerve block, which was also observed for mixtures of HXTX and VST-7304. Taken together, these data suggest that HXTX and VST-7304 have somewhat different and complementary modes of action.

Calmidazolium induces a decrease in nicotine-induced currents and intracellular calcium levels after pulse application of nicotine onto insect neurosecretory cells.

Dorsal unpaired median (DUM) neurons, are a class of insect neurosecretory cells, which are involved in the control of several functions, such as excretion and reproduction, or the release of neurohormones. Previous studies demonstrated that they express different nicotinic acetylcholine receptor subtypes, in particular α-bungarotoxin-insensitive receptors, with nAChR1 and nAChR2 subtypes. Here, we demonstrated that pulse application of 1 mM nicotine (300 ms pulse duration) induced inward currents which were reduced under bath application of 15 µM calmidazolium, a calmodulin inhibitor. Bath application of 0.5 µM α-bungarotoxin had no effect on calmidazolium action, suggesting that it could have an indirect effect through α-bungarotoxin-insensitive receptors. Indeed, nicotine-evoked currents were reduced by 10 µM d-tubocurarine, and completely blocked by 5 µM mecamylamine, which affected nAChR1 and nAChR2 subtypes, respectively. Our results demonstrated that nAChR2 subtypes are involved in the indirect effect of calmidazolium. Moreover, we found that this calmidazolium effect was associated to a strong reduction in intracellular calcium levels after pulse application of 1 mM nicotine. Thus, compared to previous studies on mammalian cells, calmidazolium did not cause an increase in intracellular calcium levels in DUM neurons, suggesting that different calcium mechanisms are involved in the calmidazolium effect.

Chemical Synthesis of a Functional Fluorescent-Tagged α-Bungarotoxin.

α-bungarotoxin is a large, 74 amino acid toxin containing five disulphide bridges, initially identified in the venom of Bungarus multicinctus snake. Like most large toxins, chemical synthesis of α-bungarotoxin is challenging, explaining why all previous reports use purified or recombinant α-bungarotoxin. However, only chemical synthesis allows easy insertion of non-natural amino acids or new chemical functionalities. Herein, we describe a procedure for the chemical synthesis of a fluorescent-tagged α-bungarotoxin. The full-length peptide was designed to include an alkyne function at the amino-terminus through the addition of a pentynoic acid linker. Chemical synthesis of α-bungarotoxin requires hydrazide-based coupling of three peptide fragments in successive steps. After completion of the oxidative folding, an azide-modified Cy5 fluorophore was coupled by click chemistry onto the toxin. Next, we determined the efficacy of the fluorescent-tagged α-bungarotoxin to block acetylcholine (ACh)-mediated currents in response to muscle nicotinic receptor activation in TE671 cells. Using automated patch-clamp recordings, we demonstrate that fluorescent synthetic α-bungarotoxin has the expected nanomolar affinity for the nicotinic receptor. The blocking effect of fluorescent α-bungarotoxin could be displaced by incubation with a 20-mer peptide mimicking the α-bungarotoxin binding site. In addition, TE671 cells could be labelled with fluorescent toxin, as witnessed by confocal microscopy, and this labelling was partially displaced by the 20-mer competitive peptide. We thus demonstrate that synthetic fluorescent-tagged α-bungarotoxin preserves excellent properties for binding onto muscle nicotinic receptors.

Interactions of Nereistoxin and Its Analogs with Vertebrate Nicotinic Acetylcholine Receptors and Molluscan ACh Binding Proteins.

Nereistoxin (NTX) is a marine toxin isolated from an annelid worm that lives along the coasts of Japan. Its insecticidal properties were discovered decades ago and this stimulated the development of a variety of insecticides such as Cartap that are readily transformed into NTX. One unusual feature of NTX is that it is a small cyclic molecule that contains a disulfide bond. In spite of its size, it acts as an antagonist at insect and mammalian nicotinic acetylcholine receptors (nAChRs). The functional importance of the disulfide bond was assessed by determining the effects of inserting a methylene group between the two sulfur atoms, creating dimethylaminodithiane (DMA-DT). We also assessed the effect of methylating the NTX and DMA-DT dimethylamino groups on binding to three vertebrate nAChRs. Radioligand receptor binding experiments were carried out using washed membranes from rat brain and fish (Torpedo) electric organ; [3H]-cytisine displacement was used to assess binding to the predominantly high affinity alpha4beta2 nAChRs and [125I]-alpha-bungarotoxin displacement was used to measure binding of NTX and analogs to the alpha7 and skeletal muscle type nAChRs. While the two quaternary nitrogen analogs, relative to their respective tertiary amines, displayed lower α4ß2 nAChR binding affinities, both displayed much higher affinities for the Torpedo muscle nAChR and rat alpha7 brain receptors than their respective tertiary amine forms. The binding affinities of DMA-DT for the three nAChRs were lower than those of NTX and MeNTX. An AChBP mutant lacking the C loop disulfide bond that would potentially react with the NTX disulfide bond displayed an NTX affinity very similar to the parent AChBP. Inhibition of [3H]-epibatidine binding to the AChBPs was not affected by exposure to NTX or MeNTX for up to 24 hr prior to addition of the radioligand. Thus, the disulfide bond of NTX is not required to react with the vicinal disulfide in the AChBP C loop for inhibition of [3H]-epibatidine binding. However, a reversible disulfide interchange reaction of NTX with nAChRs might still occur, especially under reducing conditions. Labeled MeNTX, because it can be readily prepared with high specific radioactivity and possesses relatively high affinity for the nAChR-rich Torpedo nAChR, would be a useful probe to detect and identify any nereistoxin adducts.

Evidence of a dual mechanism of action underlying the anti-proliferative and cytotoxic effects of ammonium-alkyloxy-stilbene-based α7- and α9-nicotinic ligands on glioblastoma cells.

Glioblastomas (GBMs), the most frequent brain tumours, are highly invasive and their prognosis is still poor despite the use of combination treatment. MG624 is a 4-oxystilbene derivative that is active on α7- and α9-containing neuronal nicotinic acetylcholine receptor (nAChR) subtypes. Hybridisation of MG624 with a non-nicotinic resveratrol-derived pro-oxidant mitocan has led to two novel compounds (StN-4 and StN-8) that are more potent than MG624 in reducing the viability of GBM cells, but less potent in reducing the viability of mouse astrocytes. Functional analysis of their activity on α7 receptors showed that StN-4 is a silent agonist, whereas StN-8 is a full antagonist, and neither alters intracellular [Ca2+] levels when acutely applied to U87MG cells. After 72 h of exposure, both compounds decreased U87MG cell proliferation, and pAKT and oxphos ATP levels, but only StN-4 led to a significant accumulation of cells in phase G1/G0 and increased apoptosis. One hour of exposure to either compound also decreased the mitochondrial and cytoplasmic ATP production of U87MG cells, and this was not paralleled by any increase in the production of reactive oxygen species. Knocking down the α9 subunit (which is expressed at relatively high levels in U87MG cells) decreased the potency of the effects of both compounds on cell viability, but cell proliferation, ATP production, pAKT levels were unaffected by the presence of the noncell-permeable α7/α9-selective antagonist αBungarotoxin. These last findings suggest that the anti-tumoral effects of StN-4 and StN-8 on GBM cells are not only due to their action on nAChRs, but also to other non-nicotinic mechanisms.

The balanced microenvironment regulated by the degradants of appropriate PLGA scaffolds and chitosan conduit promotes peripheral nerve regeneration.

Tissue-engineered nerve grafts (TENGs) are the most promising way for repairing long-distance peripheral nerve defects. Chitosan and poly (lactic-co-glycolic acid) (PLGA) scaffolds are considered as the promising materials in the pharmaceutical and biomedical fields especially in the field of tissue engineering. To further clarify the effects of a chitosan conduit inserted with various quantity of poly (lactic-co-glycolic acid) (PLGA) scaffolds, and their degrades on the peripheral nerve regeneration, the chitosan nerve conduit inserted with different amounts of PLGA scaffolds were used to repair rat sciatic nerve defects. The peripheral nerve regeneration at the different time points was dynamically and comprehensively evaluated. Moreover, the influence of different amounts of PLGA scaffolds on the regeneration microenvironment including inflammatory response and cell state were also revealed. The modest abundance of PLGA is more instrumental to the success of nerve regeneration, which is demonstrated in terms of the structure of the regenerated nerve, reinnervation of the target muscle, nerve impulse conduction, and overall function. The PLGA scaffolds aid the migration and maturation of Schwann cells. Furthermore, the PLGA and chitosan degradation products in a correct ratio neutralize, reducing the inflammatory response and enhancing the regeneration microenvironment. The balanced microenvironment regulated by the degradants of appropriate PLGA scaffolds and chitosan conduit promotes peripheral nerve regeneration. The findings represent a further step towards programming TENGs construction, applying polyester materials in regenerative medicine, and understanding the neural regeneration microenvironment.

Antiproliferative effect of GTS-21 in glioblastoma cells.

Glioblastoma multiforme (GBM) is the most common malignant brain tumour in adults. The poor prognosis and short median overall survival of patients with GBM is associated with resistance to therapy after surgical and adjuvant treatment. The expression of various acetylcholine receptors (AChR) in GBM has been widely reported. The present study aimed to investigate the expression of cholinergic system-related genes in primary GBM and to explore the antiproliferative effect of 3-(2,4-dimethoxybenzylidene) anabaseine (GTS-21) in GBM cell lines. Therefore, the expression of 28 genes associated with the cholinergic system was detected using a customized RT2 Profiler PCR Array in 44 GBM and 5 healthy control brain tissue samples. In addition, the activity of GTS-21, an alpha 7 subunit nicotinic AChR (α7 nAChR) agonist, and that of α-bungarotoxin (α-BTX), an α7 nAChR antagonist, was determined in primary and established GBM cells. Therefore, the A172, U87 and G28 cell lines and primary GBM cells were treated with GTS-21, ACh or nicotine. Cell viability was evaluated using MTT assay at 24, 48 and 72 h following cell treatment with the corresponding compounds. The results revealed that the expression of cholinergic system-related components was notably downregulated, except that of cholinergic receptor nicotinic alpha 7 subunit (CHRNA7), in primary GBM and U87 cells. However, the dominant-negative duplicate form of CHRNA7 was also downregulated. Furthermore, A172 and G28 cells exhibited a heterogeneous gene expression pattern. Additionally, GTS-21 inhibited the proliferation of GBM cells in a dose- and time-dependent manner. Interestingly, treatment with α-BTX restored the proliferation of U87 cells, but not that of A172 and G28 cells. Collectively, the findings of the present study suggested that GTS-21 may inhibit the proliferation of GBM cells and may therefore serve as a novel therapeutic approach to the treatment of GBM, which warrants further investigation.

Complex target SELEX-based identification of DNA aptamers against Bungarus caeruleus venom for the detection of envenomation using a paper-based device.

Complex target SELEX always have been an intriguing approach to the scientific community, as it offers the potential discovery of novel biomarkers. We herein successfully performed SELEX on Bungarus caeruleus venom to develop a panel of highly affine aptamers that specifically recognizes the B. caeruleus (common krait) venom and was able to discriminate the B. caeruleus venom from Cobra, Russell's, and Saw-scaled viper's venom. The aptamers generated against the crude venom also lead to the identification of the specific component of the venom, which is ß-Bungarotoxin, a toxin uniquely present in the B. caeruleus venom. The best performing aptamer candidates were used as a molecular recognition element in a paper-based device and were able to detect as low as 2 ng krait venom in human serum background. The developed aptamer-based paper device can be used for potential point-of-care venom detection applications due to its simplicity and affordability.

Snake C-type lectin-like proteins inhibit nicotinic acetylcholine receptors.

Venoms of viperid snakes affect mostly hemostasis, while C-type lectin-like proteins (CTLPs), one of the main components of viperid venoms, act as anticoagulants, procoagulants, or agonists/antagonists of platelet activation. However, we have shown earlier that CTLPs from the saw-scaled viper Echis multisquamatus, called emunarecins EM1 and EM2, were able to inhibit nicotinic acetylcholine receptors (nAChRs) in neurons of a pond snail (Lymnaea stagnalis). Here we analysed the structure of the emunarecins by mass spectrometry and report that EM1 and EM2 inhibit fluorescent α-bungarotoxin binding to both muscle-type nAChRs from Torpedo californica and human neuronal α7 nAChRs. EM1 at 23µM and EM2 at 9µM almost completely prevented fluorecsent α-bungarotoxin binding to muscle-type nAChRs. Interaction with human neuronal α7 nAChR was weaker; EM1 at the concentration of 23µM blocked the α-bungarotoxin binding only by about 40% and EM2 at 9µM by about 20%. The efficiency of the EM2 interaction with nAChRs was comparable to that of a non-conventional toxin, WTX, from Naja kaouthia cobra venom. Together with the data obtained earlier, these results show that CTLPs may represent new nAChR ligands.

Effects of exercise training on neuromuscular junctions and their active zones in young and aged muscles.

The neuromuscular junction (NMJ) connects the motor neuron with myofibers allowing muscle contraction. Both aging and increased activity result in NMJ remodeling. Here, the effects of exercise were examined in young and aged soleus muscles. Using immunofluorescent staining procedures, cellular and active zone components of the NMJ were quantified following a treadmill running program. Immunofluorescence was employed to determine myofiber profiles (size and type). Two-way analysis of variance procedures with main effects of age and treatment showed that when analyzing NMJs at the cellular level, significant (p ≤ 0.05) effects were identified for age, but not treatment. However, when examining subcellular active zones, effects for exercise, but not for age, were detected. Myofiber cross-sectional area showed that aging elicited atrophy and that among younger muscles endurance exercise training yielded decrements in myofiber size. Conversely, among aged muscles training elicited whole muscle and myofiber trends (p < 0.10) toward hypertrophy. Thus, different components of the neuromuscular system harbor unique sensitivities to various stimuli enabling proper adaptations to attain optimal function under differing conditions.

Molecular, physiological and behavioral characterization of the heterozygous Df[h15q13]/+ mouse model associated with the human 15q13.3 microdeletion syndrome.

The human 15q13.3 microdeletion syndrome (DS) is caused by a heterozygous microdeletion (MD) affecting six genes: FAN1; MTMR10; TRPM1; KLF13; OTUD7A; and CHRNA7. Carriers are at risk for intellectual disability, epilepsy, autism spectrum disorder, and schizophrenia. Here we used the Df[h15q13]/+ mouse model with an orthologous deletion to further characterize molecular, neurophysiological, and behavioral parameters that are relevant to the 15q13.3 DS. First, we verified the expression and distribution of the α7 nicotinic acetylcholine receptor (nAChR), a gene product of the CHRNA7, in cortical and subcortical areas. Results revealed similar mRNA distribution pattern in wildtype (WT) and heterozygous (Het) mice, with about half the number of α7 nAChR binding sites in mutants. Hippocampal recordings showed similar input/output responses of field excitatory post-synaptic potentials and theta-burst induced long-term potentiation in WT and Het mice. Het males exhibited impaired spatial learning acquisition in the Barnes Maze. Indicative of increased seizure susceptibility, Het mice developed secondary seizures after 6-Hz corneal stimulation, and had significantly increased sensitivity to the chemoconvulsant pentylenetetrazol resulting in increased spiking in hippocampal EEG recordings. Basal mRNA expression of brain derived neurotrophic factor and activity regulated immediate early genes (c-fos, Arc, Erg-1 and Npas4) during adolescence, a critical period of brain maturation, was unaffected by genotype. Thus, the MD did not show gross neuroanatomical, molecular, and neurophysiological abnormalities despite deficits in spatial learning and increased susceptibility to seizures. Altogether, our results verify the phenotypic profile of the heterozygous Df[h15q13]/+ mouse model and underscore its translational relevance for human 15q13.3 DS.

Behavioural and pharmacological profiles of zebrafish administrated pyrrolidinyl benzodioxanes and prolinol aryl ethers with high affinity for heteromeric nicotinic acetylcholine receptors.

RATIONALE: Prolinol aryl ethers and their rigidified analogues pyrrolidinyl benzodioxanes have a high affinity for mammalian α4ß2 nicotinic acetylcholine receptors (nAChRs). Electrophysiological studies have shown that the former are full agonists and the latter partial agonists or antagonists of human α4ß2 receptors, but their in vivo effects are unknown. OBJECTIVES AND METHODS: As α4ß2 nAChRs play an important role in the cognition and the rewarding effects of nicotine, we tested the effects of two full agonists and one antagonist on spatial learning, memory and attention in zebrafish using a T-maze task and virtual object recognition test (VORT). The effect of a partial agonist in reducing nicotine-induced conditioned place preference (CPP) was also investigated. RESULTS: In comparison with the vehicle alone, the full agonists MCL-11 and MCL-28 induced a significant cognitive enhancement as measured by the reduced running time in the T-maze and increased attention as measured by the increased discrimination index in the VORT. MCL-11 was 882 times more potent than nicotine. The two compounds were characterised by an inverted U-shaped dose-response curve, and their effects were blocked by the co-administration of the antagonist MCL-117, which alone had no effect. The partial agonist MCL-54 induced CPP and had an inverted U-shaped dose-response curve similar to that of nicotine but blocked the reinforcing effect of co-administered nicotine. Binding studies showed that all of the compounds have a higher affinity for heteromeric [3H]-epibatidine receptors than [125I]-αBungarotoxin receptors. MCL-11 was the most selective of heteromeric receptors. CONCLUSIONS: These behavioural studies indicate that full agonist prolinol aryl ethers are very active in increasing spatial learning, memory and attention in zebrafish. The benzodioxane partial agonist MCL-54 reduced nicotine-induced CPP, and the benzodioxane antagonist MCL-117 blocked all agonist-induced activities.