Emmanuelle Girard
Research interests
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InterestsElectrophysiology, Neurobiology
Publications
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4.52Impact points
A new mouse model for the slow-channel congenital myasthenic syndrome induced by the AChR εL221F mutation.
Neurobiology of disease. 12/2011; 45(3):851-61.
We have generated a new mouse model for congenital myasthenic syndromes by inserting the missense mutation L221F into the ε subunit of the acetylcholine receptor by homologous recombination. This mutation has been identified in man to cause a mild form of slow-channel congenital myasthenic syndrome ... [more] We have generated a new mouse model for congenital myasthenic syndromes by inserting the missense mutation L221F into the ε subunit of the acetylcholine receptor by homologous recombination. This mutation has been identified in man to cause a mild form of slow-channel congenital myasthenic syndrome with variable penetrance. In our mouse model we observe as in human patients prolonged endplate currents. The summation of endplate potentials may account for a depolarization block at increasing stimulus frequencies, moderate reduced muscle strength and tetanic fade. Calcium and intracellular vesicle accumulation as well as junctional fold loss and organelle degeneration underlying a typical endplate myopathy, were identified. Moreover, a remodeling of neuromuscular junctions occurs in a muscle-dependent pattern expressing variable phenotypic effects. Altogether, this mouse model provides new insight into the pathophysiology of congenital myasthenia and serves as a new tool for deciphering signaling pathways induced by excitotoxicity at peripheral synapses.
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2.13Impact points
The marine polyether gambierol enhances muscle contraction and blocks a transient K(+) current in skeletal muscle cells.
Toxicon : official journal of the International Society on Toxinology. 10/2010; 56(5):785-91.
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 ... [more] 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.
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3.57Impact points
Distinct localization of collagen Q and PRiMA forms of acetylcholinesterase at the neuromuscular junction.
Molecular and cellular neurosciences. 09/2010; 46(1):272-81.
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, bu... [more] 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.
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2.46Impact points
Contributions of selective knockout studies to understanding cholinesterase disposition and function.
Chemico-biological interactions. 02/2010; 187(1-3):72-7.
The complete knockout of the acetylcholinesterase gene (AChE) in the mouse yielded a surprising phenotype that could not have been predicted from deletion of the cholinesterase genes in Drosophila, that of a living, but functionally compromised animal. The phenotype of this animal showed a sufficien... [more] The complete knockout of the acetylcholinesterase gene (AChE) in the mouse yielded a surprising phenotype that could not have been predicted from deletion of the cholinesterase genes in Drosophila, that of a living, but functionally compromised animal. The phenotype of this animal showed a sufficient compromise in motor function that precluded precise characterization of central and peripheral nervous functional deficits. Since AChE in mammals is encoded by a single gene with alternative splicing, additional understanding of gene expression might be garnered from selected deletions of the alternatively spliced exons. To this end, transgenic strains were generated that deleted exon 5, exon 6, and the combination of exons 5 and 6. Deletion of exon 6 reduces brain AChE by 93% and muscle AChE by 72%. Deletion of exon 5 eliminates AChE from red cells and the platelet surface. These strains, as well as knockout strains that selectively eliminate the AChE anchoring protein subunits PRiMA or ColQ (which bind to sequences specified by exon 6) enabled us to examine the role of the alternatively spliced exons responsible for the tissue disposition and function of the enzyme. In addition, a knockout mouse was made with a deletion in an upstream intron that had been identified in differentiating cultures of muscle cells to control AChE expression. We found that deletion of the intronic regulatory region in the mouse essentially eliminated AChE in muscle and surprisingly from the surface of platelets. The studies generated by these knockout mouse strains have yielded valuable insights into the function and localization of AChE in mammalian systems that cannot be approached in cell culture or in vitro.
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3.97Impact points
Influence of MT7 toxin on the oligomerization state of the M1 muscarinic receptor.
Biology of the cell / under the auspices of the European Cell Biology Organization. 02/2010; 102(7):409-20.
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 asse... [more] 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). We analysed the hM1 oligomerization state in membrane preparations or in live cells and observed the effect of MT7 via four complementary techniques: native-PAGE electrophoresis analysed by both Western blotting and autoradiography on solubilized membrane preparations of CHO-M1 cells (Chinese-hamster ovary cells expressing muscarinic M1 receptors); FRET (fluorescence resonance energy transfer) experiments on cells expressing differently tagged M1 receptors using either an acceptor photobleaching approach or a novel fluorescence emission anisotropy technique; and, finally, by BRET (bioluminescence resonance energy transfer) assays. Our results reveal that MT7 seems to protect the M1 receptor from the dissociating effect of the detergent and induces an increase in the FRET and BRET signals, highlighting its ability to affect the dimeric form of the receptor. Our results suggest that MT7 binds to a dimeric form of hM1 receptor, favouring the stability of this receptor state at the cellular level, probably by inducing some conformational rearrangements of the pre-existing muscarinic receptor homodimers.
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5.64Impact points
Evidence for inhibition of cholinesterases in insect and mammalian nervous systems by the insect repellent deet.
BMC biology. 09/2009; 7(1):47.
ABSTRACT: BACKGROUND: N,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 deetin public health p... [more] ABSTRACT: BACKGROUND: N,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 deetin 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. RESULTS: By 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. CONCLUSION: These 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.
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4.00Impact points
The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity.
Journal of neurochemistry. 12/2008; 107(4):952-63.
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 ... [more] 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 alpha7 nAChR expressed in Xenopus oocytes. Competition-binding assays confirmed that GYM-A is a powerful ligand interacting with muscle-type nAChR, heteropentameric alpha3beta2, alpha4beta2, and chimeric alpha7-5HT(3) 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.
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7.39Impact points
Evidence of a dosage effect and a physiological endplate acetylcholinesterase deficiency in the first mouse models mimicking Schwartz-Jampel syndrome neuromyotonia.
Human molecular genetics. 10/2008; 17(20):3166-79.
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 ... [more] 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.
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7.39Impact points
A mouse model for congenital myasthenic syndrome due to MuSK mutations reveals defects in structure and function of neuromuscular junctions.
Human molecular genetics. 09/2008;
In the muscle-specific tyrosine kinase receptor gene MUSK a heteroallelic missense and a null mutation were identified in a patient suffering from a congenital myasthenic syndrome. We generated one mouse line carrying the homozygous missense mutation V789M in musk (musk(V789M/V789M) mice) and a seco... [more] In the muscle-specific tyrosine kinase receptor gene MUSK a heteroallelic missense and a null mutation were identified in a patient suffering from a congenital myasthenic syndrome. We generated one mouse line carrying the homozygous missense mutation V789M in musk (musk(V789M/V789M) mice) and a second hemizygous line, resembling the patient genotype, with the V789M mutation on one allele and an allele lacking the kinase domain (musk(V789M/-)mice). We report here that musk(V789M/V789M) mice present no obvious abnormal phenotype regarding weight, muscle function, and viability. In contrast, adult musk(V789M/-) mice suffer from severe muscle weakness, exhibit shrinkage of pelvic and scapular regions, and hunchback. Musk(V789M/-) diaphragm develops less force upon direct or nerve-induced stimulation. A profound tetanic fade is observed following nerve-evoked muscle contraction and fatigue resistance is severely impaired upon a train of tetanic nerve stimulations. Electrophysiological measurements indicate that fatigable muscle weakness is due to impaired neurotransmission as observed in a patient suffering from a congenital myasthenic syndrome. The diaphragm of adult musk(V789M/-) mice exhibits pronounced changes in endplate architecture, distribution and innervation pattern. Thus, the missense mutation V789M in MuSK acts as a hypomorphic mutation and leads to insufficiency in MuSK function in musk(V789M/-) mutants. These mutant mice represent valuable models for elucidating the roles of MuSK for synapse formation, maturation, and maintenance as well as for studying the pathophysiology of a congenital myasthenic syndrome due to MuSK mutations.
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2.56Impact points
Butyrylcholinesterase and the control of synaptic responses in acetylcholinesterase knockout mice.
Life sciences. 06/2007; 80(24-25):2380-5.
At the neuromuscular junction (NMJ) acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can hydrolyze acetylcholine (ACh). Released ACh quanta are known to diffuse rapidly across the narrow synaptic cleft and pairs of ACh molecules cooperate to open endplate channels. During their diffusion... [more] At the neuromuscular junction (NMJ) acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can hydrolyze acetylcholine (ACh). Released ACh quanta are known to diffuse rapidly across the narrow synaptic cleft and pairs of ACh molecules cooperate to open endplate channels. During their diffusion through the cleft, or after being released from muscle nicotinic ACh receptors (nAChRs), most ACh molecules are hydrolyzed by AChE highly concentrated at the NMJ. Advances in mouse genomics offered new approaches to assess the role of specific cholinesterases involved in synaptic transmission. AChE knockout mice (AChE-KO) provide a valuable tool for examining the complete abolition of AChE activity and the role of BChE. AChE-KO mice live to adulthood, and exhibit an increased sensitivity to BChE inhibitors, suggesting that BChE activity facilitated their survival and compensated for AChE function. Our results show that BChE is present at the endplate region of wild-type and AChE-KO mature muscles. The decay time constant of focally recorded miniature endplate currents was 1.04 +/- 0.06 ms in wild-type junctions and 5.4 ms +/- 0.3 ms in AChE-KO junctions, and remained unaffected by BChE-specific inhibitors, indicating that BChE is not limiting ACh duration on endplate nAChRs. Inhibition of BChE decreased evoked quantal ACh release in AChE-KO NMJs. This reduction in ACh release can explain the greatest sensitivity of AChE-KO mice to BChE inhibitors. BChE is known to be localized in perisynaptic Schwann cells, and our results strongly suggest that BChE's role at the NMJ is to protect nerve terminals from an excess of ACh.
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3.42Impact points
Brain-derived neurotrophic factor facilitates in vivo internalization of tetanus neurotoxin C-terminal fragment fusion proteins in mature mouse motor nerve terminals.
The European journal of neuroscience. 10/2006; 24(6):1546-54.
In a previous study it was reported that fusion proteins composed of the atoxic C-terminal fragment of tetanus toxin (TTC) and green fluorescent protein or beta-galactosidase (GFP-TTC and beta-gal-TTC, respectively) rapidly cluster at motor nerve terminals of the mouse neuromuscular junction (NMJ). ... [more] In a previous study it was reported that fusion proteins composed of the atoxic C-terminal fragment of tetanus toxin (TTC) and green fluorescent protein or beta-galactosidase (GFP-TTC and beta-gal-TTC, respectively) rapidly cluster at motor nerve terminals of the mouse neuromuscular junction (NMJ). Because this traffic involves presynaptic activity, probably via the secretion of active molecules, we examined whether it is affected by brain-derived neurotrophic factor (BDNF). Quantitative confocal microscopy and a fluorimetric assay for beta-gal activity revealed that co-injecting BDNF and the fusion proteins significantly increased the kinetics and amount of the proteins' localization at the NMJ and their internalization by motor nerve terminals. The observed increases were independent of synaptic vesicle recycling because BDNF did not affect spontaneous quantal acetylcholine release. In addition, injecting anti-BDNF antibody shortly before injecting GFP-TTC, and before co-injecting GFP-TTC and BDNF, significantly reduced the fusion protein's localization at the NMJ. Co-injecting GFP-TTC with neurotrophin-4 (NT-4) or glial-derived neurotrophic factor (GDNF), but not with nerve growth factor, neurotrophin-3 or ciliary neurotrophic factor, also significantly increased the fusion protein's localization at the NMJ. Thus, TTC probes may use for their neuronal internalization endocytic pathways normally stimulated by BDNF, NT-4 and GDNF binding. Different tyrosine kinase receptors with similar signalling pathways are activated by BDNF/NT-4 and GDNF binding. Thus, activated components of these signalling pathways may be involved in the TTC probes' internalization, perhaps by facilitating localization of receptors of TTC in specific membrane microdomains or by recruiting various factors needed for internalization of TTC.
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2.72Impact points
Remodeling of the neuromuscular junction in mice with deleted exons 5 and 6 of acetylcholinesterase.
Journal of molecular neuroscience : MN. 02/2006; 30(1-2):99-100.
At the vertebrate skeletal neuromuscular junction (NMJ), two closely related enzymes can hydrolyze acetylcholine (ACh): acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Advances in mouse genomics offer new approaches to assess the role of specific cholinesterases involved in neuromuscul... [more] At the vertebrate skeletal neuromuscular junction (NMJ), two closely related enzymes can hydrolyze acetylcholine (ACh): acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Advances in mouse genomics offer new approaches to assess the role of specific cholinesterases involved in neuromuscular transmission (Minic et al., 2003). AChE knockout mice provide a valuable tool for examining the effects of long-term complete and selective abolition of AChE activity (Xie et al., 2000). AChE and BChE genes encode two functional domains--the catalytic domain (exons 2, 3, and 4 of AChE, or exon 2 of BChE) and a C-terminal domain (exon 5 or 6 of AChE, or exon 3 of BChE)--that dictate the targeting of the enzymes (Massoulié, 2002). In mammals, the AChE gene produces three types of coding regions by deleting 5'- splice acceptor sites, which generate proteins; these proteins possess the same catalytic domain associated with distinct C-terminal peptides. AChE subunits of type R (readthrough) produce soluble monomers; they are expressed during development and are thought to be induced in the mouse brain by stress (Kaufer et al., 1998). AChE subunits of type H (hydrophobic) produce GPI-anchored dimers, mainly in blood cells. Subunits of type T (tailed) exist for both AChE and BChE. They represent the predominant AChE variant expressed in cholinergically innervated tissues (muscle and nerve). These subunits generate a variety of quaternary structures, including homomeric oligomers (monomers, dimers, tetramers), as well as hetero-oligomeric assemblies with anchoring proteins ColQ (Krejci et al., 1997) and PRiMA (Perrier et al., 2002). At the NMJ, AChE is clustered by the interaction of the coding sequence of exon 6 with ColQ (Feng et al., 1999). The deletion of exons 5 and 6 in the AChE gene transforms anchored AChE into a soluble enzyme (Camp et al., 2004). The present study was designed to evaluate neuromuscular transmission and nicotinic ACh receptor (nAChR) distribution in muscles from mutant mice with deletions of these two spliced exons (AChE-del-exons-5+6-/-).
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2.46Impact points
Synaptic remodeling at the skeletal neuromuscular junction of acetylcholinesterase knockout mice and its physiological relevance.
Chemico-biological interactions. 01/2006; 157-158:87-96.
Acute inhibition of synaptic acetylcholinesterase (AChE) is fatal to normal animals, but AChE-knockout mice (AChE-/-) expressing normal levels of butyrylcholinesterase (BChE) could live to adulthood without AChE expression. The present study was undertaken to determine whether compensatory mechanism... [more] Acute inhibition of synaptic acetylcholinesterase (AChE) is fatal to normal animals, but AChE-knockout mice (AChE-/-) expressing normal levels of butyrylcholinesterase (BChE) could live to adulthood without AChE expression. The present study was undertaken to determine whether compensatory mechanisms occur in the mutant that allow an effective neuromuscular transmission in the chronic absence of AChE. For this we evaluated neuromuscular transmission and the distribution of nicotinic acetylcholine receptors (nAChRs) and motor nerve terminals on isolated nerve-muscle preparations from AChE-/- mice. AChE-/- hemidiaphragm muscles maintained at 32 degrees C can support muscle twitches, and tetanic contractions during intermittent nerve-stimulation over a wide range of physiological frequencies, even though they develop less force, than age-matched wild-type (AChE+/+) muscles. Tetanic fade in AChE-/- muscles was temperature-sensitive and more marked at 22 degrees C than at 32 degrees C. Inhibition of BChE by tetraisopropylpyrophosphoramide (Iso-OMPA) intensified tetanic fade in AChE-/- muscles, but had no effect on AChE+/+ muscles, suggesting that BChE plays a protective role in nerve terminals. Skeletal muscles from AChE-/- mice adapted to the lack of AChE enzymatic activity by triggering a synaptic remodeling that critically occurred between the second and third week of postnatal development, during synapse elimination. In AChE-/- muscles nAChRs distributed in a smaller and fragmented surface area, that mirrored the branching pattern of motor nerve terminals. These findings indicate that the neuromuscular system exhibits a remarkable plasticity and adaptive responses to the chronic absence of AChE activity that has important consequences for the functioning of the neuromuscular junction.
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3.57Impact points
Internalization of a GFP-tetanus toxin C-terminal fragment fusion protein at mature mouse neuromuscular junctions.
Molecular and cellular neurosciences. 01/2006; 30(4):572-82.
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 reveal... [more] 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.
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[C-terminal fragment of tetanus toxin: its use in neuronal network analysis and its potential as non-viral vector]
Journal de la Société de biologie. 02/2005; 199(1):35-44.
The atoxic C-terminal fragment of tetanus neurotoxin or TTC fragment presents similar retrograde and transsynaptic properties to that of holotoxin. Detection of this fragment is easier when it is associated with a fluorescent marker or with beta-galactosidase activity by genetic fusion or chemical c... [more] The atoxic C-terminal fragment of tetanus neurotoxin or TTC fragment presents similar retrograde and transsynaptic properties to that of holotoxin. Detection of this fragment is easier when it is associated with a fluorescent marker or with beta-galactosidase activity by genetic fusion or chemical conjugation. Thus, these tracers have been used to study and analyse the synaptic connections of a neural network. In this article, we shortly review the various methods used with this aim including: injection of the fusion protein, adenovirus in vivo expression and transgenesis. Since neural activity is essential for neuronal TTC binding and internalization, the functionality of connections can be also evaluated. Moreover, modifications of the retrograde transport can be detected by using this fragment. Thus, TTC fragment is an excellent tracer to analyse the connectivity and functionality of a neural network. The TTC fragment was also soon proposed as potential therapeutic vector to transport and to deliver a biological activity or gene in a neural network. With this aim, the efficiency of a translocation domain to induce the cytosolic release of the associated activity has been evaluated. The use of the TTC fragment to target specifically a neurotrophic factor to neurons and thus avoid secondary effects has been tested with interesting results.
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Internalization of a GFP-tetanus toxin C-terminal fragment fusion protein at mature mouse neuromuscular junctions
Molecular and Cellular Neuroscience.
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 immunogold electron microscopy reveal... [more] 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 immunogold 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 in different axonal compartments than those used by cholera toxin B-subunit suggesting that these two proteins are transported by different pathways and cargos.
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Butyrylcholinesterase and the control of synaptic responses in acetylcholinesterase knockout mice
Life Sciences.
At the neuromuscular junction (NMJ) acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can hydrolyze acetylcholine (ACh). Released ACh quanta are known to diffuse rapidly across the narrow synaptic cleft and pairs of ACh molecules cooperate to open endplate channels. During their diffusion... [more] At the neuromuscular junction (NMJ) acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can hydrolyze acetylcholine (ACh). Released ACh quanta are known to diffuse rapidly across the narrow synaptic cleft and pairs of ACh molecules cooperate to open endplate channels. During their diffusion through the cleft, or after being released from muscle nicotinic ACh receptors (nAChRs), most ACh molecules are hydrolyzed by AChE highly concentrated at the NMJ. Advances in mouse genomics offered new approaches to assess the role of specific cholinesterases involved in synaptic transmission. AChE knockout mice (AChE-KO) provide a valuable tool for examining the complete abolition of AChE activity and the role of BChE. AChE-KO mice live to adulthood, and exhibit an increased sensitivity to BChE inhibitors, suggesting that BChE activity facilitated their survival and compensated for AChE function. Our results show that BChE is present at the endplate region of wild-type and AChE-KO mature muscles. The decay time constant of focally recorded miniature endplate currents was 1.04 ± 0.06 ms in wild-type junctions and 5.4 ms ± 0.3 ms in AChE-KO junctions, and remained unaffected by BChE-specific inhibitors, indicating that BChE is not limiting ACh duration on endplate nAChRs. Inhibition of BChE decreased evoked quantal ACh release in AChE-KO NMJs. This reduction in ACh release can explain the greatest sensitivity of AChE-KO mice to BChE inhibitors. BChE is known to be localized in perisynaptic Schwann cells, and our results strongly suggest that BChE's role at the NMJ is to protect nerve terminals from an excess of ACh.
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A mouse model for congenital myasthenic syndrome due to MuSK mutations reveals defects in structure and function of neuromuscular junctions
In the muscle-specific tyrosine kinase receptor gene MUSK , a heteroallelic missense and a null mutation were identified in a patient suffering from a congenital myasthenic syndrome (CMS). We generated one mouse line carrying the homozygous missense mutation V789M in musk ( musk V789M/V789M mice) an... [more] In the muscle-specific tyrosine kinase receptor gene MUSK , a heteroallelic missense and a null mutation were identified in a patient suffering from a congenital myasthenic syndrome (CMS). We generated one mouse line carrying the homozygous missense mutation V789M in musk ( musk V789M/V789M mice) and a second hemizygous line, resembling the patient genotype, with the V789M mutation on one allele and an allele lacking the kinase domain ( musk V789M/− mice). We report here that musk V789M/V789M mice present no obvious abnormal phenotype regarding weight, muscle function and viability. In contrast, adult musk V789M/− mice suffer from severe muscle weakness, exhibit shrinkage of pelvic and scapular regions and hunchback. Musk V789M/− diaphragm develops less force upon direct or nerve-induced stimulation. A profound tetanic fade is observed following nerve-evoked muscle contraction, and fatigue resistance is severely impaired upon a train of tetanic nerve stimulations. Electrophysiological measurements indicate that fatigable muscle weakness is due to impaired neurotransmission as observed in a patient suffering from a CMS. The diaphragm of adult musk V789M/− mice exhibits pronounced changes in endplate architecture, distribution and innervation pattern. Thus, the missense mutation V789M in MuSK acts as a hypomorphic mutation and leads to insufficiency in MuSK function in musk V789M/− mutants. These mutant mice represent valuable models for elucidating the roles of MuSK for synapse formation, maturation and maintenance as well as for studying the pathophysiology of a CMS due to MuSK mutations.
Following (7)
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Jordi Molgo
Le Centre national de la recherche scientifique -
Vincent Corbel
Institut de Recherche pour le Développement -
Veronique Bernard
Institut national de la santé et de la recherche médicale -
Frédérique Rene
Université de Strasbourg -
Oksana Lockridge
University of Nebraska Medical Center
Topics (3)
