Emmanuelle Girard

Evidence for inhibition of cholinesterases in insect and mammalian nervous systems by the insect repellent deet

BackgroundN,N-Diethyl-3-methylbenzamide (deet) remains the gold standard for insect repellents. About 200 million people use it every year and over 8 billion doses have been applied over the past 50 years. Despite the widespread and increased interest in the use of deet in public health programmes, controversies remain concerning both the identification of its target sites at the olfactory system and its mechanism of toxicity in insects, mammals and humans. Here, we investigated the molecular target site for deet and the consequences of its interactions with carbamate insecticides on the cholinergic system.ResultsBy using toxicological, biochemical and electrophysiological techniques, we show that deet is not simply a behaviour-modifying chemical but that it also inhibits cholinesterase activity, in both insect and mammalian neuronal preparations. Deet is commonly used in combination with insecticides and we show that deet has the capacity to strengthen the toxicity of carbamates, a class of insecticides known to block acetylcholinesterase.ConclusionThese findings question the safety of deet, particularly in combination with other chemicals, and they highlight the importance of a multidisciplinary approach to the development of safer insect repellents for use in public health.

The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity

Gymnodimines (GYMs) are phycotoxins exhibiting unusual structural features including a spirocyclic imine ring system and a trisubstituted tetrahydrofuran embedded within a 16‐membered macrocycle. The toxic potential and the mechanism of action of GYM‐A, highly purified from contaminated clams, have been assessed. GYM‐A in isolated mouse phrenic hemidiaphragm preparations produced a concentration‐ and time‐dependent block of twitch responses evoked by nerve stimulation, without affecting directly elicited muscle twitches, suggesting that it may block the muscle nicotinic acetylcholine (ACh) receptor (nAChR). This was confirmed by the blockade of miniature endplate potentials and the recording of subthreshold endplate potentials in GYM‐A paralyzed frog and mouse isolated neuromuscular preparations. Patch‐clamp recordings in Xenopus skeletal myocytes revealed that nicotinic currents evoked by constant iontophoretical ACh pulses were blocked by GYM‐A in a reversible manner. GYM‐A also blocked, in a voltage‐independent manner, homomeric human α7 nAChR expressed in Xenopus oocytes. Competition‐binding assays confirmed that GYM‐A is a powerful ligand interacting with muscle‐type nAChR, heteropentameric α3β2, α4β2, and chimeric α7‐5HT3 neuronal nAChRs. Our data show for the first time that GYM‐A broadly targets nAChRs with high affinity explaining the basis of its neurotoxicity, and also pave the way for designing specific tests for accurate GYM‐A detection in shellfish samples.

Catecholamine secretion from rat foetal adrenal chromaffin cells and hypoxia sensitivity

The adrenal medulla chromaffin cells (AMCs) secrete catecholamines in response to various types of stress. We examined the hypoxia-sensitivity of catecholamine secretion by rat foetal chromaffin cells in which the innervation by the splanchnic nerve is not established. The experiments were performed in primary cultured cells from two different ages of foetuses (F15 and F19). Membrane potential of AMCs was monitored with the patch clamp technique, and the catecholamine secretion was detected by amperometry. We found that: (1) AMCs from F19 foetuses showed hypoxia-induced catecholamine release. (2) This hypoxia-induced secretion is produced by membrane depolarization generated by an inhibition of Ca2+-activated K+ current [IK(Ca)] current. (3) Chromaffin precursor cells from F15 foetuses secrete catecholamine. The quantal release is calcium-dependent, but the size of the quantum is reduced. (4) In the precursor cells, a hypoxia-induced membrane hyperpolarization is originated by an ATP-sensitive K+ current [IK(ATP)] activation. (5) During the prenatal period, at F15, the percentage of the total outward current for IK(ATP) and IK(Ca) was 50 and 29.5%, respectively, whereas at F19, IK(ATP) is reduced to 14%, and IK(Ca) became 64% of the total current. We conclude that before birth, the age-dependent hypoxia response of chromaffin cells is modulated by the functional activity of KATP and KCa channels.

Evidence of a dosage effect and a physiological endplate acetylcholinesterase deficiency in the first mouse models mimicking Schwartz–Jampel syndrome neuromyotonia

Schwartz-Jampel syndrome (SJS) is a recessive neuromyotonia with chondrodysplasia. It results from hypomorphic mutations of the gene encoding perlecan, leading to a decrease in the levels of this heparan sulphate proteoglycan in basement membranes (BMs). It has been suggested that SJS neuromyotonia may result from endplate acetylcholinesterase (AChE) deficiency, but this hypothesis has never been investigated in vivo due to the lack of an animal model for neuromyotonia. We used homologous recombination to generate a knock-in mouse strain with one missense substitution, corresponding to a human familial SJS mutation (p.C1532Y), in the perlecan gene. We derived two lines, one with the p.C1532Y substitution alone and one with p.C1532Y and the selectable marker Neo, to down-regulate perlecan gene activity and to test for a dosage effect of perlecan in mammals. These two lines mimicked SJS neuromyotonia with spontaneous activity on electromyogramm (EMG). An inverse correlation between disease severity and perlecan secretion in the BMs was observed at the macroscopic and microscopic levels, consistent with a dosage effect. Endplate AChE levels were low in both lines, due to synaptic perlecan deficiency rather than major myofibre or neuromuscular junction disorganization. Studies of muscle contractile properties showed muscle fatigability at low frequencies of nerve stimulation and suggested that partial endplate AChE deficiency might contribute to SJS muscle stiffness by potentiating muscle force. However, physiological endplate AChE deficiency was not associated with spontaneous activity at rest on EMG in the diaphragm, suggesting that additional changes are required to generate such activity characteristic of SJS.

Isolation and pharmacological characterization of AdTx1, a natural peptide displaying specific insurmountable antagonism of the α1A-adrenoceptor

Background and purpose:  Venoms are a rich source of ligands for ion channels, but very little is known about their capacity to modulate G‐protein coupled receptor (GPCR) activity. We developed a strategy to identify novel toxins targeting GPCRs.

Internalization of a GFP-tetanus toxin C-terminal fragment fusion protein at mature mouse neuromuscular junctions.

The distribution, dynamics, internalization, and retrograde axonal traffic of a fusion protein composed of green fluorescent protein (GFP)and the atoxic C-terminal fragment of tetanus toxin (TTC) were studied after its in vivo injection. Confocal microscopy and immuno-gold electron microscopy revealed that the fusion protein (GFP-TTC) rapidly clustered in motor nerve terminals of the neuromuscular junction. Clathrin-coated pits, and axolemma infoldings located between active zones appeared to be involved in the internalization of the fusion protein. Biochemical analysis of detergent-extracted neuromuscular preparations showed that the GFP-TTC fusion protein was associated with lipid microdomains. We suggest that GFP-TTC clustering in these lipid microdomains favors the recruitment of other proteins involved in its endocytosis and internalization in motor nerve terminals. During its retrograde trafficking, GFP-TTC accumulated indifferent axonal compartments than those used by cholera toxin B-subunit suggesting that these two proteins are transported by different pathways and cargos.

MuSK frizzled-like domain is critical for mammalian neuromuscular junction formation and maintenance.

The muscle-specific kinase MuSK is one of the key molecules orchestrating neuromuscular junction (NMJ) formation. MuSK interacts with the Wnt morphogens, through its Frizzled-like domain (cysteine-rich domain [CRD]). Dysfunction of MuSK CRD in patients has been recently associated with the onset of myasthenia, common neuromuscular disorders mainly characterized by fatigable muscle weakness. However, the physiological role of Wnt-MuSK interaction in NMJ formation and function remains to be elucidated. Here, we demonstrate that the CRD deletion of MuSK in mice caused profound defects of both muscle prepatterning, the first step of NMJ formation, and synapse differentiation associated with a drastic deficit in AChR clusters and excessive growth of motor axons that bypass AChR clusters. Moreover, adult MuSKΔCRD mice developed signs of congenital myasthenia, including severe NMJs dismantlement, muscle weakness, and fatigability. We also report, for the first time, the beneficial effects of lithium chloride, a reversible inhibitor of the glycogen synthase kinase-3, that rescued NMJ defects in MuSKΔCRD mice and therefore constitutes a novel therapeutic reagent for the treatment of neuromuscular disorders linked to Wnt-MuSK signaling pathway deficiency. Together, our data reveal that MuSK CRD is critical for NMJ formation and plays an unsuspected role in NMJ maintenance in adulthood.

Distinct localization of Collagen Q and PRiMA forms of Acetylcholinesterase at the neuromuscular junction

Acetylcholinesterase (AChE) terminates the action of acetylcholine at cholinergic synapses thereby preventing rebinding of acetylcholine to nicotinic postsynaptic receptors at the neuromuscular junction. Here we show that AChE is not localized close to these receptors on the postsynaptic surface, but is instead clustered along the presynaptic membrane and deep in the postsynaptic folds. Because AChE is anchored by ColQ in the basal lamina and is linked to the plasma membrane by a transmembrane subunit (PRiMA), we used a genetic approach to evaluate the respective contribution of each anchoring oligomer. By visualization and quantification of AChE in mouse strains devoid of ColQ, PRiMA or AChE, specifically in the muscle, we found that along the nerve terminus the vast majority of AChE is anchored by ColQ that is only produced by the muscle, whereas very minor amounts of AChE are anchored by PRiMA that is produced by motoneurons. In its synaptic location, AChE is therefore positioned to scavenge ACh that effluxes from the nerve by non-quantal release. AChE-PRiMA, produced by the muscle, is diffusely distributed along the muscle in extrajunctional regions.

The marine polyether gambierol enhances muscle contraction and blocks a transient K(+) current in skeletal muscle cells.

Gambierol is a complex marine toxin first isolated with ciguatoxins from cell cultures of the toxic dinoflagellate Gambierdiscus toxicus. Despite the chemical complexity of the polycyclic ether toxin, the total successful synthesis of gambierol has been achieved by different chemical strategies. In the present work the effects of synthetic gambierol on mouse and frog skeletal neuromuscular preparations and Xenopus skeletal myocytes have been studied. Gambierol (0.1-5 muM) significantly increased isometric twitch tension in neuromuscular preparations stimulated through the motor nerve. Less twitch augmentation was observed in directly stimulated muscles when comparing twitch tension-time integrals obtained by nerve stimulation. Also, gambierol induced small spontaneous muscle contraction originating from presynaptic activity that was completely inhibited by d-tubocurarine. Gambierol slowed the rate of muscle action potential repolarization, triggered spontaneous and/or repetitive action potentials, and neither affected action potential amplitude nor overshoot in skeletal muscle fibers. These results suggest that gambierol through an action on voltage-gated K(+) channels prolongs the duration of action potentials, enhances the extent and time course of Ca(2+) release from the sarcoplasmic reticulum, and increases twitch tension generation. Further evidence is provided that gambierol at sub-micromolar concentrations blocks a fast inactivating outward K(+) current that is responsible for action potential prolongation in Xenopus skeletal myocytes.

Influence of MT7 toxin on the oligomerization state of the M1 muscarinic receptor1

Background information. The idea that GPCRs (G‐protein‐coupled receptors) may exist as homo‐ or hetero‐oligomers, although still controversial, is now widely accepted. Nevertheless, the functional roles of oligomerization are still unclear and gaining greater insight into the mechanisms underlying the dynamics of GPCR assembly and, in particular, assessing the effect of ligands on this process seems important. We chose to focus our present study on the effect of MT7 (muscarinic toxin 7), a highly selective allosteric peptide ligand, on the oligomerization state of the hM1 (human M1 muscarinic acetylcholine receptor subtype).