Article

Identification of Nicotinic Acetylcholine Receptor Recycling and Its Role in Maintaining Receptor Density at the Neuromuscular Junction In Vivo

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Abstract

In the CNS, receptor recycling is critical for synaptic plasticity; however, the recycling of receptors has never been observed at peripheral synapses. Using a novel imaging technique, we show here that nicotinic acetylcholine receptors (AChRs) recycle into the postsynaptic membrane of the neuromuscular junction. By sequentially labeling AChRs with biotin-bungarotoxin and streptavidin-fluorophore conjugates, we were able to distinguish recycled, preexisting, and new receptor pools at synapses in living mice. Time-lapse imaging revealed that recycled AChRs were incorporated into the synapse within hours of initial labeling, and their numbers increased with time. At fully functional synapses, AChR recycling was robust and comparable in magnitude with the insertion of newly synthesized receptors, whereas chronic synaptic activity blockade nearly abolished receptor recycling. Finally, using the same sequential labeling method, we found that acetylcholinesterase, another synaptic component, does not recycle. These results identify an activity-dependent AChR-recycling mechanism that enables the regulation of receptor density, which could lead to rapid alterations in synaptic efficacy.

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... Their studies laid the foundations of the secretory pathway, which is used by nAChR, and led to several principal methods, including electron microscopy, radioautography, pulse/chase labeling, and cell fractionation (reviewed in [19][20][21] the introduction of α-bungarotoxin [22,23] and its derivatives (reviewed in [24,25]), which have served as efficient tools to identify or isolate nAChR, the scene was set in the mid-1970s to address nAChR trafficking. Live animal imaging [26,27] combined with radioactive or fluorescence pulse/chase analysis and genetic or pharmacological interventions were instrumental for the understanding of kinetic and molecular aspects of nAChR turnover [10,11,[28][29][30][31][32][33][34]. Finally, genetically encoded molecular biosensors further refined the insights into the molecular machineries for nAChR trafficking [35][36][37][38][39][40][41][42]. ...
... Starting in 2005, a series of reports on nAChR trafficking used an elegant fluorescence-based pulse/chase-like labeling assay [28,29,[31][32][33]52]. These combined injection of α-bungarotoxin-biotin at one time point (here referred to as "pulse") with a sequence of immediate and later injections ("chase") of streptavidin-species marked with different fluorophores. ...
... Subsequent microscopy revealed that surface-exposed nAChR labeled by injection of α-bungarotoxin-biotin plus streptavidin-dye would (i) remain α-bungarotoxin-biotin positive unless degraded, (ii) lose their fluorescence tag upon endocytosis, and (iii) expose a free streptavidin-binding site upon recycling. One insight from these studies was that˜25% of nAChR undergo recycling within four days [28]. Notably, this was subject to extensive regulation. ...
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At vertebrate motor endplates, the conversion of nerve impulses into muscle contraction is initiated by binding of acetylcholine to its nicotinic receptor (nAChR) at the postsynapse. Efficiency and safety of this process are dependent on proper localization, density, and molecular composition of the receptors. To warrant this, intricate machineries regulating the turnover of nAChR are in place. They control and execute the processes of i) expression, ii) delivery to the postsynaptic membrane, iii) clustering at the plasma membrane, iv) endocytic retrieval, v) activity-dependent recycling, and vi) degradation of nAChR. Concentrating on aspects iv-vi, this review addresses the current status of techniques, concepts, and open questions on endocytosis, recycling, and degradation of nAChR. A picture is emerging, that shows connections between executing machineries and their regulators. The first group includes the actin cytoskeleton, myosin motor proteins, Rab G-proteins, and the autophagic cascade. The second group features protein kinases A and C, Cdk5, and CaMKII as well as other components like the E3-ligase MuRF1 and the membrane shaping regulator, SH3GLB1. Recent studies have started to shed light onto nerve inputs that appear to master the tuning of the postsynaptic protein trafficking apparatus and the expression of critical components for nAChR turnover.
... On subsequent days (typically 4-5 days later), the animals were re-anesthetized and muscles were exposed and bathed with a second saturating dose of streptavidin-Alexa 488 and BTX-Alexa 430 to label the recycled and newly synthesized AChRs respectively. Control experiments for ruling out dissociation of streptavidin from biotin on the surface of the muscle cells were described in our previous work (Bruneau et al., 2005). Muscles were removed and fixed with 4% paraformaldehyde, mounted on coverslips and scanned with a confocal microscope (Leica; model SPE) using a 100×, 1.46 numerical aperture (NA) oilimmersion objective (Leica; HCX Apochromat). ...
... To examine whether internalized AChRs were able to recycle to agrin-induced ectopic AChR clusters, we co-injected fibers of rat soleus muscle with expression plasmids for nls-GFP (allowing later identification of injected fibers) and for full length chicken agrin (NtAcagrin748) (Denzer et al., 1995) at a concentration of 1 mg/ml, i.e. a high concentration inducing clusters of relatively high AChR density. Three to four weeks later, the muscles were bathed in situ with a saturating dose of bungarotoxin-biotin (BTX-biotin) to label surface receptors, followed by a saturating dose of streptavidin-Alexa594 (red: see Figure 1) to label all biotin sites as described by Bruneau et al. (Bruneau et al., 2005). Four days later, the soleus muscle was exposed and sequentially labeled with streptavidin-Alexa488 (green) to specifically label the recycled receptor pool (receptors that had lost their initial streptavidin-Alexa594 tag while retaining BTX-biotin during the process of internalization and reinsertion in the postsynaptic membrane receptors) and BTX-Alexa430 (blue) to label newly synthesized receptors that had been inserted after the initial labeling. ...
... At innervated NMJs the recycling of receptors significantly contributes to the steady-state of the postsynaptic receptor density. However, in aneural cultured muscle cells (nerve-free) or in inactive muscles (denervated muscles or muscles blocked with pharmacological agents), the recycling of AChR is severely depressed or nonexistent (Bruneau et al., 2005;Bruneau and Akaaboune, 2006). We asked whether the nerve-derived factor agrin is sufficient to induce the recycling of AChRs, and, if so, whether this could explain the previously reported metabolic stabilization of receptors at ectopic AChRs by agrin. ...
Article
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During the development of the neuromuscular junction, motor axons induce the clustering of acetylcholine receptors (AChRs) and increase their metabolic stability in the muscle membrane. Here, we asked whether the synaptic organizer agrin might regulate the metabolic stability and density of AChRs by promoting the recycling of internalized AChRs, which would otherwise be destined for degradation, into synaptic sites. We show that at nerve-free AChR clusters induced by agrin in extrasynaptic membrane, internalized AChRs are driven back into the ectopic synaptic clusters where they intermingle with pre-existing and new receptors. The extent of AChR recycling depended on the strength of the agrin stimulus, but not on the development of junctional folds, another hallmark of mature postsynaptic membranes. In chronically denervated muscles, in which both AChR stability and recycling are significantly decreased by muscle inactivity, agrin maintained the amount of recycled AChRs at agrin-induced clusters at a level similar to that at denervated original endplates. In contrast, AChRs did not recycle at agrin-induced clusters in C2C12 or primary myotubes. Thus, in muscles in vivo, but not in cultured myotubes, neural agrin promotes the recycling of AChRs and thereby increases their metabolic stability.
... Together, our knowledge about these pathologies highlights the relevance of: a) maintaining a high density of postsynaptic nAChRs for proper NMJ function, b) the nAChR trafficking pathways, such as internalization and, more recently, recycling (Bruneau et al., 2005), in the maintenance of this density and c) the role of receptor-associated proteins in nAChR stability (Gilhus, 2012). Accordingly, this introduction will further discuss the nAChR intracellular trafficking pathways and the role of receptor-associated proteins in the regulation of receptor dynamics. ...
... For decades, it was widely believed that nAChRs were stable in the postsynaptic membrane until they were removed for degradation. Recent work from our lab has shown that, instead, many internalized nAChRs recycle back to the plasma membrane, and this 'recycled pool' contributes significantly to the steady-state of the postsynaptic receptor density (Bruneau et al., 2005;Bruneau and Akaaboune, 2006). This process has been shown to occur in an activity-dependent manner, as blocking synaptic activity depresses the recycling of receptors into synaptic sites. ...
... This process has been shown to occur in an activity-dependent manner, as blocking synaptic activity depresses the recycling of receptors into synaptic sites. However, much of the molecular machinery governing the recycling and trafficking of receptor-containing vesicles remains unknown (Bruneau et al., 2005(Bruneau et al., , 2008(Bruneau et al., , 2009Bruneau and Akaaboune, 2006;Wu et al., 2010). The discovery of recycled receptors at the NMJ has prompted us to re-evaluate the metabolic stability of receptors. ...
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The effectiveness of synaptic transmission at most mammalian synapses depends largely on the maintenance of a high density of postsynaptic receptors. In a mature synapse, this density is highly dynamic and can be regulated by several factors including synaptic activity, post-translational modifications of receptors, and scaffold proteins. In my thesis work, I focused on the regulation of AChR clustering, which is the hallmark of a neuromuscular junction, a well characterized cholinergic synapse between the motor neuron and the skeletal muscle. Among several pathways, I first focused on the role of alpha-syntrophin (syn), a member of the dystrophin glycoprotein complex (DGC), in the development and modulation of nAChR dynamics of the mouse NMJ. Using syn knock-out mice, I showed that syn is not required for synapse formation, but it is essential for synapse maturation. Particularly, I demonstrated that during the maturation of synapses, the integrity of the postsynaptic apparatus is altered, the turnover rate of AChRs increases significantly, and the number/density of AChRs is impaired. The synaptic alterations observed in this mouse mutant were explained by the loss of tyrosine phosphorylated alpha-dystrobrevin (dbn). Interestingly, when GFP-dbn1 was electroporated into sternomastoid muscles of syn mutant, most of synaptic abnormalities were partially restored. In the second part of my thesis work, I investigated the role of serine/threonine kinases, particularly PKC and PKA on the regulation of nAChR trafficking. We found that PKC accelerates nAChR removal and inhibits recycling at the NMJ, while PKA has the opposite effect. Finally, I begin to address the role of the Wnt/beta-catenin pathway in the adult NMJ, and we show that beta-catenin interacts with the DGC in mature synapses, via rapsyn. Taken together, these results provide new insights into the cellular and molecular underlying signaling of the regulation of nAChR trafficking and dynamics.
... Next, we asked whether PKC also affects the normal rate of recycling of previously internalized AChRs into the postsynaptic membrane. To this end, AChRs on the sternomastoid muscle were sequentially labeled with BTX-biotin, followed by a saturating dose of strept-Alexa488, as described in our previous work [3,24]. Four days later, recycled receptors were specifically labeled by adding (red) streptavidin-Alexa594 (strept-Alexa594) to the sternomastoid muscle (strept-Alexa594 binds to receptors that have lost their initial strept-Alexa488 tag while retaining BTX-biotin) [24]. ...
... To this end, AChRs on the sternomastoid muscle were sequentially labeled with BTX-biotin, followed by a saturating dose of strept-Alexa488, as described in our previous work [3,24]. Four days later, recycled receptors were specifically labeled by adding (red) streptavidin-Alexa594 (strept-Alexa594) to the sternomastoid muscle (strept-Alexa594 binds to receptors that have lost their initial strept-Alexa488 tag while retaining BTX-biotin) [24]. Superficial synapses were imaged immediately (time 0), and the sternomastoid muscle was then bathed with calphostin C, a highly specific PKC blocker, to inhibit PKC [25,26] for the duration of the experiment (7 hours). ...
... The fluorescence intensity of labeled recycled AChRs was measured before and after treatment. Quantification of recycled AChRs shows that after 7 hours of calphostin C treatment, the fluorescence intensity increased to 114 ± 8% (n = 57 NMJs, 7 mice) of their original fluorescence at time 0 (normalized at 100%) compared to untreated synapses where fluorescence remains unchanged, as previously described by Bruneau et al. [24] (102 ± 3%, n = 15 NMJs, p < 0.001, 3 mice) (Figure 1 E, F). As a second test of PKC inhibition, we used staurosporine (100 nM), a moderately potent PKC blocker, and found that the re-insertion of recycled AChRs at synaptic sites after 7 hours of treatment was also increased, albeit slightly less than with calphostin C (fluorescence intensity of recycled receptors was 106 ± 5% (n = 17 NMJs, 4 mice) versus untreated synapses, 99 ± 3% (n = 21 NMJs, 4 mice, p < 0.001). ...
Article
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The steady state of the acetylcholine receptor (AChR) density at the neuromuscular junction (NMJ) is critical for efficient and reliable synaptic transmission. However, little is known about signaling molecules involved in regulating the equilibrium between the removal and insertion of AChRs that establishes a stable postsynaptic receptor density over time. In this work, we tested the effect of activities of two serine/threonine kinases, PKC and PKA, on the removal rate of AChRs from and the re-insertion rate of internalized recycled AChRs into synaptic sites of innervated and denervated NMJs of living mice. Using an in vivo time-lapse imaging approach and various pharmacological agents, we showed that PKC and PKA activities have antagonistic effects on the removal and recycling of AChRs. Inhibition of PKC activity or activation of PKA largely prevents the removal of pre-existing AChRs and promotes the recycling of internalized AChRs into the postsynaptic membrane. In contrast, stimulation of PKC or inactivation of PKA significantly accelerates the removal of postsynaptic AChRs and depresses AChR recycling. These results indicate that a balance between PKA and PKC activities may be critical for the maintenance of the postsynaptic receptor density.
... In 2005, with the development of the sequential method of labeling of AChRs with biotin-bungarotoxin and streptavidin-fluorophore conjugates, it was demonstrated that a significant number of internalized AChRs were able to recycle back to synaptic sites [35,37,38]. This method allows us to distinctly visualize three distinct receptor pools at the NMJ: recycled, pre-existing, and newly synthesized receptors [37]. ...
... In 2005, with the development of the sequential method of labeling of AChRs with biotin-bungarotoxin and streptavidin-fluorophore conjugates, it was demonstrated that a significant number of internalized AChRs were able to recycle back to synaptic sites [35,37,38]. This method allows us to distinctly visualize three distinct receptor pools at the NMJ: recycled, pre-existing, and newly synthesized receptors [37]. Of note, it is well established that AChR pools have the same binding affinities for biotin-bungarotoxin/streptavidin. ...
... In the absence of muscle activity following surgical denervation or pharmacological agents-induced paralysis, the turnover rate of AChRs was found to be considerably more rapid than the turnover rate in active muscles. For instance, at surgically denervated NMJs, the half-life of AChRs was significantly reduced from ∼ 9-14 days to 1-3 days [37,[52][53][54]. A similar increase in the turnover of AChRs was observed in long-term inactivity of the innervated muscle caused by a tetrodotoxin cuff on the nerve [55]. ...
Article
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The clustering and maintenance of nicotinic acetylcholine receptors (AChRs) at high density in the postsynaptic membrane is a hallmark of the mammalian neuromuscular junction (NMJ). The regulation of receptor density/turnover rate at synapses is one of the main thrusts of neurobiology because it plays an important role in synaptic development and synaptic plasticity. The state-of-the-art imaging revealed that AChRs are highly dynamic despite the overall structural stability of the NMJ over the lifetime of the animal. This review highlights the work on the metabolic stability of AChRs at developing and mature NMJs and discusses the role of synaptic activity and the regulatory signaling pathways involved in the dynamics of AChRs.
... On their removal from the synapse, a fraction of the internalized AChRs becomes reinserted into the synaptic membrane (recycled AChRs; Bruneau et al., 2005). To examine whether LL5β knockdown differentially affects the rate of insertion of newly made and recycled AChRs, we stained and imaged AChRs as outlined in Figure 3D. ...
... (described in the Constructs section of Supplemental Methods) was electroporated in mouse sternomastoid muscle as described and analyzed 2-3 wk later. To determine AChR density at the synapse, the sternomastoid muscle was labeled for 1 h with 5 μg/ml α-BTX-Alexa 594 (a dose that has been demonstrated to be sufficient to saturate all receptors; Bruneau et al., 2005), and superficial synapses were imaged. Only fibers in which synaptic nuclei were marked by Histone2B-RFP, indicative of either shLL5β or shScrambled expression, were analyzed. ...
... AChR insertion was assessed from the recovery of GFP fluorescence in clusters of GFP-AChRs upon photobleaching a circled region of To determine the removal rate of the different AChR pools (preexisting and recycled AChRs), the sternomastoid muscle of anesthetized mice was exposed to α-BTX-biotin (1 μg/ml, 10 min), followed by a single saturating dose of streptavidin-Alexa 488, so that synaptic transmission remained functional (Akaaboune et al., 2002), and synapses were imaged. Three days later, preexisting receptors from the same synapses were imaged, following which, recycled receptors that had lost the streptavidin-Alexa 488 in the process of recycling (Bruneau et al., 2005) and were reinserted into the synaptic membrane were labeled with streptavidin-Alexa 488 and reimaged. Finally, newly added receptors were labeled with a saturating dose of BTX-biotin/streptavidin-Alexa 488 and imaged. ...
Article
A hallmark of the neuromuscular junction (NMJ) is the high density of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane. The postsynaptic apparatus of the NMJ is organized by agrin secreted from motor neurons. The mechanisms that underlie the focal delivery of AChRs to the adult NMJ are not yet understood in detail. We previously showed that microtubule (MT) capture by the plus-end tracking protein CLASP2 regulates AChR density at agrin-induced AChR clusters in cultured myotubes via PI3 kinase acting through GSK3β. Here, we show that knock-down of the CLASP2-interaction partner LL5β by RNAi and forced expression of a CLASP2 fragment blocking the CLASP2/LL5β interaction inhibit microtubule capture. The same treatments impair focal vesicle delivery to the clusters. Consistent with these findings, knock-down of LL5β at the NMJ in vivo reduces the density and insertion of AChRs into the postsynaptic membrane. MT capturing and focal vesicle delivery to agrin-induced AChR clusters are also inhibited by microtubule and actin depolymerizing drugs, invoking both cytoskeletal systems in MT capture and in the fusion of AChR vesicles with the cluster membrane. Combined our data identify a transport system, organized by agrin through PI3 kinase, GSK3β, CLASP2 and LL5β, for precise delivery of AChR vesicles from the subsynaptic nuclei to the overlying synaptic membrane. © 2015 by The American Society for Cell Biology.
... 20 Injected into live animals, BGT labels surface-exposed postsynaptic CHRN and allows to study endo/lysosomal and recycling processes of CHRN. 19,21,22 Imaging of CHRN labeled with BGT-AlexaFluor647 (BGT-AF647) in live mouse tibialis anterior (TA) muscles, showed a steady-state amount of »9 endocytic CHRN vesicles per NMJ. 22 Overexpression of a GTPase-deficient hyperactive Q79L mutant of RAB5, a major regulator of early endosomal steps, 23 amplified the number of CHRN puncta to »520. ...
... In the same period, 20% of CHRN recycle and 20% of CHRN are newly made to replenish the NMJ. 19 Accordingly, one can assume that 20% of CHRN are degraded within a 24 h period, also fitting to data from BGT-radioiodine approaches. 5,35 Additionally, RAB5 overexpression was found to enhance CHRN degradation rate, 22 and homotypic fusion of endocytic vesicles 24 likely contributed to a further reduction in the amount of BGT-positive puncta after 24 h. ...
Article
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Vertebrate skeletal muscle contraction is mediated by nicotinic acetylcholine receptors (CHRN). Endocytosis and recycling of CHRN regulate their proper abundance at nerve-muscle synapses, i.e. neuromuscular junctions. Recent work showed that RAB5 is essential for CHRN endocytosis. Here, using in vivo-imaging of endocytosed CHRN and RAB-GFP fusion proteins, we deliver evidence for differential effects of RAB5-GFP, RAB4-GFP, and RAB11-GFP on CHRN endocytosis. Furthermore, while newly endocytosed CHRN colocalized with RAB5-GFP over large stretches of muscle fibers, RAB4-GFP and RAB11-GFP colocalized with endocytosed CHRN almost exclusively at neuromuscular junctions. In agreement with previous findings, this data suggests the existence of a specialized subsynaptic zone that is particularly relevant for CHRN recycling.
... Thus, 12-20% of AChR density must already have been lost at the start of the efgartigimod treatment on the fifth passive transfer day, and this loss will likely have progressed for a number of days, in spite of lowered MuSK IgG4 levels. 3) Recovery from this loss depends on insertion of new AChRs, a process that takes a few days (Bruneau et al., 2005). Furthermore, AChR density is at dynamic equilibrium with degradation compensated for by insertion of newly synthesized plus recycling of existing AChRs (Bruneau et al., 2005). ...
... 3) Recovery from this loss depends on insertion of new AChRs, a process that takes a few days (Bruneau et al., 2005). Furthermore, AChR density is at dynamic equilibrium with degradation compensated for by insertion of newly synthesized plus recycling of existing AChRs (Bruneau et al., 2005). Both these mechanisms may be disturbed at the MuSK MG NMJ, hampering recovery. ...
Article
Myasthenia gravis is hallmarked by fatigable muscle weakness resulting from neuromuscular synapse dysfunction caused by IgG autoantibodies. The variant with muscle-specific kinase (MuSK) autoantibodies is characterized by prominent cranial and bulbar weakness and a high frequency of respiratory crises. The majority of MuSK MG patients requires long-term immunosuppressive treatment, but the result of these treatments is considered less satisfactory than in MG with acetylcholine receptor antibodies. Emergency treatments are more frequently needed, and many patients develop permanent facial weakness and nasal speech. Therefore, new treatment options would be welcome. The neonatal Fc receptor protects IgG from lysosomal breakdown, thus prolonging IgG serum half-life. Neonatal Fc receptor antagonism lowers serum IgG levels and thus may act therapeutically in autoantibody-mediated disorders. In MuSK MG, IgG4 anti-MuSK titres closely correlate with disease severity. We therefore tested efgartigimod (ARGX-113), a new neonatal Fc receptor blocker, in a mouse model for MuSK myasthenia gravis. This model involves 11 daily injections of purified IgG4 from MuSK myasthenia gravis patients, resulting in overt myasthenic muscle weakness and, consequently, body weight loss. Daily treatment with 0.5 mg efgartigimod, starting at the fifth passive transfer day, reduced the human IgG4 titres about 8-fold, despite continued daily injection. In muscle strength and fatigability tests, efgartigimod-treated myasthenic mice outperformed control myasthenic mice. Electromyography in calf muscles at endpoint demonstrated less myasthenic decrement of compound muscle action potentials in efgartigimod-treated mice. These substantial in vivo improvements of efgartigimod-treated MuSK MG mice following a limited drug exposure period were paralleled by a tendency of recovery at neuromuscular synaptic level (in various muscles), as demonstrated by ex vivo functional studies. These synaptic improvements may well become more explicit upon longer drug exposure. In conclusion, our study shows that efgartigimod has clear therapeutic potential in MuSK myasthenia gravis and forms an exciting candidate drug for many autoantibody-mediated neurological and other disorders.
... At the mature NMJs, it has long been believed that AChRs are metabolically stable in the postsynaptic membrane at the mature NMJs, until they are internalized and targeted to lysosomes for protein degradation [45]. In fact, some internalized AChRs are recycled and inserted back to the postsynaptic membrane for maintaining a high postsynaptic AChR density [24,46]. Using biotin-α-BTX and streptavidin conjugates of different fluorophores, it is possible to distinguish various pools of AChRs, namely preexisting, recycling, and newly synthesized AChRs [46,47]. ...
... In fact, some internalized AChRs are recycled and inserted back to the postsynaptic membrane for maintaining a high postsynaptic AChR density [24,46]. Using biotin-α-BTX and streptavidin conjugates of different fluorophores, it is possible to distinguish various pools of AChRs, namely preexisting, recycling, and newly synthesized AChRs [46,47]. This sequential labeling approach will allow us to investigate the trafficking mechanisms of endocytosed AChR vesicles following endocytosis. ...
Article
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Purpose of Review At present, the most common diagnostic measures for the autoimmune neuromuscular disease myasthenia gravis (MG) are radioimmunoprecipitation assay (RIPA), enzyme-linked immunosorbent assay (ELISA), and cell-based assay (CBA). Considering the pitfalls of these diagnostic assays, this review describes the advantages of using Xenopus tissue cultures for MG diagnosis and research. Recent Findings Our recent study described a novel CBA involving Xenopus tissue cultures for MG serological diagnosis. Moreover, this CBA can potentially be applied to elucidate the pathogenic mechanisms underlying acetylcholine receptor endocytosis and degradation and to develop and validate potential therapeutic strategies for MG. Summary Although most CBAs are relatively labor intensive, Xenopus CBA is a promising tool for the initial clinical serological diagnosis and for the pathological research of MG. The future studies will be devoted to gain a better understanding of the etiology of MG and to provide a therapeutic intervention for this disease.
... Third, studies with chimeric foetal/adult receptors have shown that foetal nAChRs determine a proper distribution and innervation pattern of nascent NMJs (Cetin et al., 2020). Fourth, nAChR subunit replacement may also be related to postsynaptic stability, as the half-life of nAChRs increases as NMJ maturation proceeds (Bruneau & Akaaboune, 2006;Bruneau et al., 2005). ...
... In this regard, it has been described that nAChRs are internalised through an endocytic pathway that depends on Rac1 but is independent of clathrin, caveolin and dynamin (Kumari et al., 2008). Furthermore, it has been reported that nAChRs into low-density areas are not stable (Bruneau & Akaaboune, 2006;Bruneau et al., 2005) as they are rapidly internalised and redistributed within nAChR highdensity areas of pretzels (Lee et al., 2009) ('nAChR degradation' in Figure 1c). Interestingly, this process is controlled by the actindepolymerising factor/cofilin (Lee et al., 2009) which, along with the podosome initiation markers cortactin and the Arp2/3 complex (Millard et al., 2004;Webb et al., 2006) ECM remodelling, which could alter nAChR stability in the sarcolemma, also favouring pretzel formation. ...
Article
The neuromuscular junction (NMJ) is the peripheral synapse formed between a motor axon and a skeletal muscle fibre that allows muscle contraction and the coordinated movement in many species. A main hallmark of the mature NMJ is the assembly of nicotinic acetylcholine receptor (nAChR) aggregates in the muscle postsynaptic domain, that distributes in perfect apposition to presynaptic motor terminals. To assemble its unique functional architecture, initial embryonic NMJs undergo an early postnatal maturation process characterised by the transformation of homogenous nAChR-containing plaques to elaborate and branched pretzel-like structures. In spite of a detailed morphological characterisation, the molecular mechanisms controlling the intracellular scaffolding that organises a postsynaptic domain at the mature NMJ have not been fully elucidated. In this review, we integrate evidence of key processes and molecules that have shed light on our current understanding of the NMJ maturation process. On the one hand, we consider in vitro studies revealing the potential role of podosome-like structures to define discrete low nAChR-containing regions to consolidate a plaque-to-pretzel transition at the NMJ. On the other hand, we focus on in vitro and in vivo evidence demonstrating that members of the Ras homologous (Rho) protein family of small GTPases (small Rho GTPases) play indispensable roles on NMJ maturation by regulating the stability of nAChR aggregates. We combine this evidence to propose that small Rho GTPases are key players in the assembly of podosome-like structures that drive the postsynaptic maturation of vertebrate NMJs.
... Subsequent fates include storage in an intracellular compartment, recycling to the post-synaptic membrane in an activity-dependent manner, and degradation (Figure 5). Given that about 25% of all surface receptors are normally recycled within 4 days (Bruneau et al., 2005), positive or negative tuning of the decision between recycling and deterioration would also affect AChR density at the NMJ, and if recycling is lacking spatial precision, this could potentially lead to fragmentation of synapses due to receptor delivery at wrong sites. A couple of reports indicate that second messenger-triggered serine/threonine phosphorylation events are major determinants controlling the decision-making between recycling and degradation . ...
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Functional denervation is a hallmark of aging sarcopenia as well as of muscular dystrophy. It is thought to be a major factor reducing skeletal muscle mass, particularly in the case of sarcopenia. Neuromuscular junctions (NMJs) serve as the interface between the nervous and skeletal muscular systems, and thus they may receive pathophysiological input of both pre- and post-synaptic origin. Consequently, NMJs are good indicators of motor health on a systemic level. Indeed, upon sarcopenia and dystrophy, NMJs morphologically deteriorate and exhibit altered characteristics of primary signaling molecules, such as nicotinic acetylcholine receptor and agrin. Since a remarkable reversibility of these changes can be observed by exercise, there is significant interest in understanding the molecular mechanisms underlying synaptic deterioration upon aging and dystrophy and how synapses are reset by the aforementioned treatments. Here, we review the literature that describes the phenomena observed at the NMJ in sarcopenic and dystrophic muscle as well as to how these alterations can be reversed and to what extent. In a second part, the current information about molecular machineries underlying these processes is reported.
... Fluorophore-conjugated natural antagonists have also been utilized, with in many cases the highly potent peptidic snake toxin a-bungarotoxin (a-BgTx) conjugated to a wide range of different fluorophores including rhodamine, fluorescein isothiocyanate (FITC), Alexa dyes or biotin, for use in immunofluorescence studies with streptavidin-linked fluorophores (Anderson and Cohen, 1974;Baier et al., 2010;Bruneau et al., 2005;Qu et al., 1990;Wheeler et al., 1994). Other natural peptidic antagonists used in fluorescent studies include a-cobratoxin and conotoxins (Hone et al., 2009;Johnson et al., 1990;Yang et al., 2011). ...
... The fate of the AChRs lost from postsynaptic AChR clusters remains uncertain. At healthy NMJs in vivo a fraction of the endplate AChRs become internalized into an endosome-like compartment before being returned to the postsynaptic membrane (Bruneau et al. 2005). This fraction of recycling AChRs had particularly short half-lives at the endplate (Bruneau & Akaaboune, 2006), suggesting that they tend to be diverted to lysosomal degradation (the eventual fate of AChRs; Hyman & Froehner, 1983;Christianson & Green, 2004). ...
Article
Muscle Specific Kinase (MuSK) autoantibodies from myasthenia gravis patients can block the activation of MuSK in vitro and/or reduce the postsynaptic localization of MuSK. Here we use a mouse model to examine the effects of MuSK autoantibodies upon some key components of the postsynaptic MuSK pathway and upon the regulation of junctional ACh receptor (AChR) numbers. Mice became weak after 14 daily injections of anti-MuSK-positive patient IgG. The intensity and area of AChR staining at the motor endplate was markedly reduced. Pulse labelling of AChRs revealed an accelerated loss of pre-existing AChRs from postsynaptic AChR clusters without a compensatory increase in incorporation of (newly-synthesized) replacement AChRs. Large, postsynaptic AChR clusters were replaced by a constellation of tiny AChR microaggregates. Puncta of AChR staining also appeared in the cytoplasm beneath the endplate. Endplate staining for MuSK, activated Src, rapsyn and AChR were all reduced in intensity. In the tibialis anterior muscle there was also evidence that phosphorylation of the AChR β-subunit-Y390 was reduced at endplates. In contrast, endplate staining for β-dystroglycan (through which rapsyn couples AChR to the synaptic basement membrane) remained intense. The results suggest that anti-MuSK IgG suppresses the endplate density of MuSK, thereby down-regulating MuSK signalling activity and the retention of junctional AChRs locally within the postsynaptic membrane scaffold.This article is protected by copyright. All rights reserved
... Thus, to display the vesicles containing endocytosed CHRN along with their corresponding NMJs, post-acquisition contrast enhancement was used, similar to previous studies of this and other laboratories. 16,18,25,26 Quantitative analysis revealed that denervation alone led to more than an 8-fold increase in the internalized CHRN vesicle number (Fig. 1A-B and E). Chloroquine treatment seemingly blocked the processing of these vesicles and led to their significant enrichment in innervated as well as denervated muscles by a factor of 2-3 when compared to the same condition in the absence of chloroquine ( Fig. 1C-E). ...
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Endocytosed nicotinic acetylcholine receptors (CHRN) are degraded via macroautophagy/autophagy during atrophic conditions and are accompanied by the autophagic regulator protein SH3GLB1. The present study addressed the functional role of SH3GLB1 on CHRN trafficking and its implementation. We found an augmented ratio of total SH3GLB1 to threonine-145 phosphorylated SH3GLB1 (SH3GLB1:p-SH3GLB1) under conditions of increased CHRN vesicle numbers. Overexpression of T145 phosphomimetic (T145E) and phosphodeficient (T145A) mutants of SH3GLB1, were found to either slow down or augment the processing of endocytic CHRN vesicles, respectively. Co-expression of the early endosomal orchestrator RAB5 largely rescued the slow processing of endocytic CHRN vesicles induced by T145E. SH3GLB1 phosphomutants did not modulate the expression or colocalization of RAB5 with CHRN vesicles, but instead altered the expression of RAB5 activity regulators. In summary, these findings suggest that SH3GLB1 controls CHRN endocytic trafficking in a phosphorylation- and RAB5-dependent manner at steps upstream of autophagosome formation.
... La quantité de RnACh à la membrane post-synaptique reste en général constante grâce à des mécanismes d'exocytose et d'endocytose fonctionnant en permanence. Ainsi, les nouveaux RnACh seraient ajoutés par exocytose et les anciens RnACh éliminés par endocytose dans la zone péri-synaptique, selon le modèle proposé par Sanes et Lichtman, les replis synaptiques pouvant également fournir un itinéraire par lequel les RnACh pourraient entrer ou sortir des zones denses ( Figure 12) Marchand et al., 2002;Bruneau et al., 2005;Bruneau and Akaaboune, 2006). ...
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ColQ is a specific collagen that anchors acetylcholinesterase (AChE) in the synaptic cleft of the neuromuscular junction. The importance of AChE-ColQ complex in the physiology of this synapse has been highlighted by the identification of COLQ mutations in the human gene, leading to a congenital myasthenic syndrome with AChE deficiency (CMS-1c). The symptoms observed in patients and the defects of the neuromuscular junctions in adult ColQ mutant mice which are mice model for this CMS-1c are complex and AChE deficiency cannot account for all of them. We hypothesized that ColQ could play a role per se in synapse formation and therefore its absence would participate in the defects. I have shown that ColQ regulates the levels of nicotinic acetylcholine receptor (nAChR) subunits via MuSK, a key protein in synapse formation, independently of AChE. The consequence of this regulation is that ColQ controls nAChR clustering implicated in synaptic transmission. Moreover, my results have shown that ColQ deficiency reduces membrane-bound MuSK. In this context, it is highly significant that the phenotypes of CMS-1c and MuSK CMS patients have similarities. In addition, atrophic signs were found in ColQ-/- mice muscles suggesting a partial denervation of muscle fibers in the absence of ColQ. Modifications highlighted by my results may thus explain the origin of the functional defects linked to ColQ absence or mutation.
... The metabolic stability of the synaptic AChR is not determined by the rate of their internalization alone. Rather, upon their internalization, a fraction of the synaptic AChRs is targeted to degradation, whereas another fraction is recycled to the synaptic membrane (61). Thus the measured metabolic AChR half-life is determined by the net result of the rates of AChR internalization, the fractions of AChRs that are recycled versus degraded after internalization. ...
Article
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The neuromuscular junction is the chemical synapse between motor neurons and skeletal muscle fibers. It is designed to reliably convert the action potential from the presynaptic motor neuron into the contraction of the postsynaptic muscle fiber. Diseases that affect the neuromuscular junction may cause failure of this conversion and result in loss of ambulation and respiration. The loss of motor input also causes muscle wasting as muscle mass is constantly adapted to contractile needs by the balancing of protein synthesis and protein degradation. Finally, neuromuscular activity and muscle mass have a major impact on metabolic properties of the organisms. This review discusses the mechanisms involved in the development and maintenance of the neuromuscular junction, the consequences of and the mechanisms involved in its dysfunction, and its role in maintaining muscle mass during aging. As life expectancy is increasing, loss of muscle mass during aging, called sarcopenia, has emerged as a field of high medical need. Interestingly, aging is also accompanied by structural changes at the neuromuscular junction, suggesting that the mechanisms involved in neuromuscular junction maintenance might be disturbed during aging. In addition, there is now evidence that behavioral paradigms and signaling pathways that are involved in longevity also affect neuromuscular junction stability and sarcopenia. Copyright © 2015 the American Physiological Society.
... With such a method it would be possible to image two different proteins in the same cell, or in neighboring cells, each with single molecule sensitivity. Additionally, although the biotin-streptavidin binding affinity is very high, the off-rate increases 10-fold or more when each is conjugated to a macromolecule, leading to dissociation on the order of hours, 6,7 or on the order of seconds with applied force. 8,9 We were therefore also motivated to develop a covalent targeting method for long-term imaging applications. ...
Article
We present a methodology for targeting quantum dots to specific proteins on living cells in two steps. In the first step, Escherichia coli lipoic acid ligase (LplA) site-specifically attaches 10-bromodecanoic acid onto a 13 amino acid recognition sequence that is genetically fused to a protein of interest. In the second step, quantum dots derivatized with HaloTag, a modified haloalkane dehalogenase, react with the ligated bromodecanoic acid to form a covalent adduct. We found this targeting method to be specific, fast, and fully orthogonal to a previously reported and analogous quantum dot targeting method using E. coli biotin ligase and streptavidin. We used these two methods in combination for two-color quantum dot visualization of different proteins expressed on the same cell or on neighboring cells. Both methods were also used to track single molecules of neurexin, a synaptic adhesion protein, to measure its lateral diffusion in the presence of neuroligin, its trans-synaptic adhesion partner.
... We also detected certain BBQcontaining fluorescent bodies that were smaller than the aforementioned puncta and were not colocalized with EEA1 (Fig. 1E, arrowheads); these presumably were surface AChRs or newly internalized AChRs that had not fused with endosomes. At mature NMJs, the recycling of endocytosed AChRs back to the muscle surface has been identified using a well-established sequentiallabeling protocol [25]. Because of the broad excitation spectra of the 2 kinds of QDs used in this study [26], we could not accurately determine the fractions of endocytosed AChRs in recycling versus lysosomal pathways by using the previously published sequentiallabeling protocol that involves labeling with biotin-BTX and streptavidin-QD conjugates. ...
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In a majority of patients with myasthenia gravis (MG), anti-acetylcholine receptor (AChR) antibodies target postsynaptic AChR clusters and thus compromise the membrane integrity of neuromuscular junctions (NMJs) and lead to muscle weakness. Antibody-induced endocytosis of AChRs in the postsynaptic membrane represents the initial step in the pathogenesis of MG; however, the molecular mechanisms underlying AChR endocytosis remain largely unknown. Here, we developed an approach to mimic the pathogenic antibodies for inducing the crosslinking and internalization of AChRs from the postsynaptic membrane. Using biotin-α-bungarotoxin and quantum dot (QD)-streptavidin, cell-surface and internalized AChRs could be readily distinguished by comparing the size, fluorescence intensity, trajectory, and subcellular localization of the QD signals. QD-induced AChR endocytosis was mediated by clathrin-dependent and caveolin-independent mechanisms, and the trafficking of internalized AChRs in the early endosomes required the integrity of microtubule structures. Furthermore, activation of the agrin/MuSK (muscle-specific kinase) signaling pathway strongly suppressed QD-induced internalization of AChRs. Lastly, QD-induced AChR crosslinking potentiated the dispersal of aneural AChR clusters upon synaptic induction. Taken together, our results identify a novel approach to study the mechanisms of AChR trafficking upon receptor crosslinking and endocytosis, and demonstrate that agrin-MuSK signaling pathways protect against crosslinking-induced endocytosis of AChRs.
... Indeed, newly formed AChRs are assembled in the ER and then transported through Golgi apparatus and exocytic vesicles to the postsynaptic membrane, involving the assistance of the receptor-associated protein rapsyn (Marchand et al. 2002). Subsequently, receptors get endocytosed (Fumagalli et al. 1982;Engel and Fumagalli 1982) and then are either recycled to the plasma membrane (Bruneau et al. 2005) or get degraded via an autophagosomal-lysosomal pathway (Rudolf et al. 2013;Khan et al. 2014). Several signaling mechanisms involved in the regulation of AChR turnover have been unraveled and methods to address AChR turnover in live model organisms are of continued interest in the quest for treating neuromuscular disorders. ...
Article
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The turnover of nicotinic acetylcholine receptors (AChR) is a critical factor that determines function and safety of neuromuscular transmission at the nerve-muscle synapses, i.e. neuromuscular junctions (NMJs). Previously, three different populations of AChRs exhibiting distinct stereotypic and activity-dependent half-life values were observed in mouse muscles. To address AChR turnover in more detail, we here employed a recently developed longitudinal radioiodine assay that is based on repetitive measurements of radio emission from the same animals over long periods of time in combination with systematic variation of the time elapsed between AChR pulse-labeling and muscle denervation. Modeling of the data revealed profiles of AChR de novo synthesis and receptor incorporation into the postsynaptic membrane. Furthermore, decay of pre-existing AChRs upon denervation showed a peculiar pattern corroborating earlier findings of a two-step stabilization of AChRs.
... In the presence of shear force in a flowing environment, lower binding affinity to streptavidin is observed even with free, non-conjugated biotin moieties [32]. Furthermore, high rates of dissociation for biotinylated biomolecules from streptavidin have been reported under the low pH conditions of the endosome [33]. Therefore, for many practical applications, there is a need to have stronger interactions. ...
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To extend and improve the utility of the streptavidin-binding peptide tag (SBP-tag) in applications ranging from affinity purification to the reversible immobilization of recombinant proteins, a cysteine residue was introduced to the streptavidin mutein SAVSBPM18 and the SBP-tag to generate SAVSBPM32 and SBP(A18C), respectively. This pair of derivatives is capable of forming a disulfide bond through the newly introduced cysteine residues. SAVSBPM32 binds SBP-tag and biotin with binding affinities (Kd ~ 10-8M) that are similar to SAVSBPM18. Although SBP(A18C) binds to SAVSBPM32 more weakly than SBP-tag, the binding affinity is sufficient to bring the two binding partners together efficiently before they are locked together via disulfide bond formation-a phenomenon we have named affinity-driven thiol coupling. Under the condition with SBP(A18C) tags in excess, two SBP(A18C) tags can be captured by a tetrameric SAVSBPM32. The stoichiometry of the disulfide-bonded SAVSBPM32-SBP(A18C) complex was determined using a novel two-dimensional electrophoresis method which has general applications for analyzing the composition of disulfide-bonded protein complexes. To illustrate the application of this reversible immobilization technology, optimized conditions were established to use the SAVSBPM32-affinity matrix for the purification of a SBP(A18C)-tagged reporter protein to high purity. Furthermore, we show that the SAVSBPM32-affinity matrix can also be applied to purify a biotinylated protein and a reporter protein tagged with the unmodified SBP-tag. The dual (covalent and non-covalent) binding modes possible in this system offer great flexibility to many different applications which need reversible immobilization capability.
... Moreover, in line with the idea of NMJ formation in these cultures, a portion of the neurons expressed a non-phosphorylated epitope in neurofilament H (SMI-32), a motoneuron marker ( Figure 4D). Finally, when NMJs were labeled with fluorogenic α-bungarotoxin, a specific marker of nicotinic acetylcholine receptors (nAChR) known to be densely expressed in the NMJ postsynaptic membrane (Bruneau et al., 2005), bright fluorescence was observed at end plates ( Figure 4E) and colocalized with synaptophysin immunoreactivity (Figure 4F). These results led us to hypothesize that both the neuronal cells and the postsynaptic membranes present in myotubes of our human co-culture model are mature enough to form functional NMJs. ...
Article
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Several neuromuscular diseases involve dysfunction of neuromuscular junctions (NMJs), yet there are no patient-specific human models for electrophysiological characterization of NMJ. We seeded cells of neurally-induced embryoid body-like spheres derived from induced pluripotent stem cell (iPSC) or embryonic stem cell (ESC) lines as monolayers without basic fibroblast factor (bFGF) and observed differentiation of neuronal as well as spontaneously contracting, multinucleated skeletal myotubes. The myotubes showed striation, immunoreactivity for myosin heavy chain, actin bundles typical for myo-oriented cells, and generated spontaneous and evoked action potentials (APs). The myogenic differentiation was associated with expression of MyoD1, myogenin and type I ryanodine receptor. Neurons formed end plate like structures with strong binding of α-bungarotoxin, a marker of nicotinic acetylcholine receptors highly expressed in the postsynaptic membrane of NMJs, and expressed SMI-32, a motoneuron marker, as well as SV2, a marker for synapses. Pharmacological stimulation of cholinergic receptors resulted in strong depolarization of myotube membrane and raised Ca2+ concentration in sarcoplasm, while electrical stimulation evoked Ca2+ transients in myotubes. Stimulation of motoneurons with N-Methyl-D-aspartate resulted in reproducible APs in myotubes and end plates displayed typical mEPPs and tonic activity depolarizing myotubes of about 10 mV. We conclude that simultaneous differentiation of neurons and myotubes from patient-specific iPSCs or ESCs results also in the development of functional NMJs. Our human model of NMJ may serve as an important tool to investigate normal development, mechanisms of diseases and novel drug targets involving NMJ dysfunction and degeneration.
... CHRN are bona fide transmembrane proteins and are composed of 5 subunits that are synthesized and assembled at the level of the endoplasmic reticulum, before they reach the postsynapstic membrane via Golgi apparatus and exocytic vesicles. 11,12 After their arrival at the membrane, CHRN can either persist there or get endocytosed in vesicular carriers 13,14 from where it might be either recycled back to the membrane or get degraded [15][16][17] in lysosomes. 18 While the expression of CHRN displays strong upregulation under muscle wasting conditions, 19,20 such as immobilization and denervation, its metabolic stability decreases at the same time, 10,21-23 implicating post-transcriptional regulation in CHRN turnover under stress. ...
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Removal of ubiquitinated targets by autophagosomes can be mediated by receptor molecules, like SQSTM1, in a mechanism referred to as selective autophagy. While cytoplasmic protein aggregates, mitochondria, and bacteria are the best-known targets of selective autophagy, their role in the turnover of membrane receptors is scarce. We here showed that fasting-induced wasting of skeletal muscle involves remodeling of the neuromuscular junction (NMJ) by increasing the turnover of muscle-type CHRN (cholinergic receptor, nicotinic/nicotinic acetylcholine receptor) in a TRIM63-dependent manner. Notably, this process implied enhanced production of endo/lysosomal carriers of CHRN, which also contained the membrane remodeler SH3GLB1, the E3 ubiquitin ligase, TRIM63, and the selective autophagy receptor SQSTM1. Furthermore, these vesicles were surrounded by the autophagic marker MAP1LC3A in an ATG7-dependent fashion, and some of them were also positive for the lysosomal marker, LAMP1. While the amount of vesicles containing endocytosed CHRN strongly augmented in the absence of ATG7 as well as upon denervation as a model for long-term atrophy, denervation-induced increase in autophagic CHRN vesicles was completely blunted in the absence of TRIM63. On a similar note, in trim63(-/-) mice denervation-induced upregulation of SQSTM1 and LC3-II was abolished and endogenous SQSTM1 did not colocalize with CHRN vesicles as it did in the wild type. SQSTM1 and LC3-II coprecipitated with surface-labeled/endocytosed CHRN and SQSTM1 overexpression significantly induced CHRN vesicle formation. Taken together, our data suggested that selective autophagy regulates the basal and atrophy-induced turnover of the pentameric transmembrane protein, CHRN, and that TRIM63, together with SH3GLB1 and SQSTM1 regulate this process.
... The amino terminal domain has a beta sheet structure that is involved in assembly of the pentameric complex, and the transmembrane segments are alpha helices that should be aligned to form a hydrophilic ion pore 30 . Maintaining density of nAChR in the synapse is important for regulating effective communication between neurons, and significant variation of these receptors and the recycling mechanism affects brain plasticity 31 . Through electron microscopy and electrophysiology techniques, the structure and transition states of the receptor in its various conformations have been discovered as follows: closed (in the absence of ligand), open (in the presence of ligand), and desensitized (where there is a high affinity for ligand) 32 . ...
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Background: Nicotine addiction is a complex and multifactorial disease affecting the central nervous system and consists of a set of characteristic symptoms and signs. Objective: The objective of this study was to provide an overview on smoking and the complexity of dependency, with special emphasis on the involvement of genetic factors, including neurexin and nicotinic cholinergic receptor genes. Methods: The following two aspects are discussed in the present article: (i) epidemiology in Mexico; and (ii) a review of the published literature on genetic association studies using the National Center for Biotechnology Information (NCBI) database of the USA as a search tool. The search key words were: nicotine, smoking, dependence, genetic, tobacco, neurobiology and GWAS. The publication period of the reviewed articles was January 2005 to July 2015. Results: There are numerous studies that provide evidence of the involvement of a genetic component that contributes to the risk of developing nicotine addiction, but the multifactorial nature of addiction requires coordinated research from multiple disciplines. Conclusion: Research is needed on the factors associated with genetic risk for nicotine addiction and their interaction with environmental factors.
... Importantly, despite obvious similarities, NKA and GLUT4 are not localized in the same intracellular vesicles (196), indicating that membrane abundance of different membrane proteins is regulated by distinct regulatory pathways. In addition to NKA and GLUT4, dynamic translocation to and from the membrane has also been demonstrated for amino acid transporters (143), transferrin receptor (200), receptor for low-density lipoproteins (168), and even large macromolecular complexes like the nicotinic acetylcholine receptor (nAChR) (42,222). Clearly, muscle fibers can dynamically regulate abundance of various membrane proteins by controlling their subcellular localization. ...
Article
Skeletal muscle contains one of the largest and the most dynamic pools of Na,K-ATPase (NKA) in the body. Under resting conditions NKA in skeletal muscle operates at only a fraction of maximal pumping capacity, but it can be markedly activated when demands for ion transport increase, such as during exercise or following food intake. Given the size, capacity, and dynamic range of the NKA pool in skeletal muscle, its tight regulation is essential to maintain whole-body homeostasis as well as muscle function. To reconcile functional needs of systemic homeostasis with those of skeletal muscle, NKA is regulated in a coordinated manner by extrinsic stimuli, such as hormones and nerve-derived factors, as well as by local stimuli arising in skeletal muscle fibers, such as contractions and muscle energy status. These stimuli regulate NKA acutely by controlling its enzymatic activity and/or its distribution between plasma membrane and the intracellular storage compartment. They also regulate NKA chronically by controlling NKA gene expression, thus determining total NKA content in skeletal muscle and its maximal pumping capacity. This review focuses on molecular mechanisms that underlie regulation of NKA in skeletal muscle by major extrinsic and local stimuli. Special emphasis is given to stimuli and mechanisms linking regulation of NKA and energy metabolism in skeletal muscle, such as insulin and the energy-sensing AMP-activated protein kinase. Finally, the recently uncovered roles for glutathionylation, nitric oxide, and extracellular K(+) in regulation of NKA in skeletal muscle are highlighted.
... Using different elegant labeling approaches with the highly AChR-selective snake venom, α-bungarotoxin, Engel et al. showed by electron microscopy that AChR is endocytosed in membrane-bound carriers (Engel et al., 1977;Fumagalli et al., 1982) and several groups established an activity-dependent metabolic stabilization of AChR (Fambrough, 1979;Levitt et al., 1980;Loring and Salpeter, 1980;Levitt and Salpeter, 1981;Drachman, 1981, 1983;Salpeter and Loring, 1985;Shyng et al., 1991;Salpeter, 1997, 1999). Next, Akaaboune et al. demonstrated that AChR is recycled to the postsynaptic membrane in an activity-dependent manner (Akaaboune et al., 1999;Bruneau et al., 2005;Bruneau and Akaaboune, 2006). At this point a large part of the lifecycle of AChRs was described phenomenologically. ...
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Autonomic regulation processes in striated muscles are largely mediated by cAMP/PKA-signaling. In order to achieve specificity of signaling its spatial-temporal compartmentation plays a critical role. We discuss here how specificity of cAMP/PKA-signaling can be achieved in skeletal muscle by spatio-temporal compartmentation. While a microdomain containing PKA type I in the region of the neuromuscular junction (NMJ) is important for postsynaptic, activity-dependent stabilization of the nicotinic acetylcholine receptor (AChR), PKA type I and II microdomains in the sarcomeric part of skeletal muscle are likely to play different roles, including the regulation of muscle homeostasis. These microdomains are due to specific A-kinase anchoring proteins, like rapsyn and myospryn. Importantly, recent evidence indicates that compartmentation of the cAMP/PKA-dependent signaling pathway and pharmacological activation of cAMP production are aberrant in different skeletal muscles disorders. Thus, we discuss here their potential as targets for palliative treatment of certain forms of dystrophy and myasthenia. Under physiological conditions, the neuropeptide, α-calcitonin-related peptide, as well as catecholamines are the most-mentioned natural triggers for activating cAMP/PKA signaling in skeletal muscle. While the precise domains and functions of these first messengers are still under investigation, agonists of β2-adrenoceptors clearly exhibit anabolic activity under normal conditions and reduce protein degradation during atrophic periods. Past and recent studies suggest direct sympathetic innervation of skeletal muscle fibers. In summary, the organization and roles of cAMP-dependent signaling in skeletal muscle are increasingly understood, revealing crucial functions in processes like nerve-muscle interaction and muscle trophicity.
... It is beyond the scope of the present study to determine the precise mechanisms that underlie the distinctly different kinetics of restoring (a3b4) 2 a5vs. (a3b4) 2 b4-nAChR surface expression after PI-PLC cleavage of the lynx1 GPI anchor; however, complete restoration within 60 min may suggest a role for a rapid (a3b4) 2 a5-nAChR internalization and recycling mechanism, as has been demonstrated for both neuromuscular and nonmuscle nAChR subtypes (60)(61)(62)(63). This observation reinforces the previous indication that lynx1-induced reductions in I max can primarily be attributed to a reduction in the number of cell-surface receptors. ...
Article
This study investigates-for the first time to our knowledge-the existence and mechanisms of functional interactions between the endogenous mammalian prototoxin, lynx1, and α3- and β4-subunit-containing human nicotinic acetylcholine receptors (α3β4*-nAChRs). Concatenated gene constructs were used to express precisely defined α3β4*-nAChR isoforms (α3β4)2β4-, (α3β4)2α3-, (α3β4)2α5(398D)-, and (α3β4)2α5(398N)-nAChR in Xenopus oocytes. In the presence or absence of lynx1, α3β4*-nAChR agonist responses were recorded by using 2-electrode voltage clamp and single-channel electrophysiology, whereas radioimmunolabeling measured cell-surface expression. Lynx1 reduced (α3β4)2β4-nAChR function principally by lowering cell-surface expression, whereas single-channel effects were primarily responsible for reducing (α3β4)2α3-nAChR function [decreased unitary conductance (≥50%), altered burst proportions (3-fold reduction in the proportion of long bursts), and enhanced closed dwell times (3- to 6-fold increase)]. Alterations in both cell-surface expression and single-channel properties accounted for the reduction in (α3β4)2α5-nAChR function that was mediated by lynx1. No effects were observed when α3β4*-nAChRs were coexpressed with mutated lynx1 (control). Lynx1 is expressed in the habenulopeduncular tract, where α3β4*-α5*-nAChR subtypes are critical contributors to the balance between nicotine aversion and reward. This gives our findings a high likelihood of physiologic significance. The exquisite isoform selectivity of lynx1 interactions provides new insights into the mechanisms and allosteric sites [α(-)-interface containing] by which prototoxins can modulate nAChR function.-George, A. A., Bloy, A., Miwa, J. M., Lindstrom, J. M., Lukas, R. J., Whiteaker, P. Isoform-specific mechanisms of α3β4*-nicotinic acetylcholine receptor modulation by the prototoxin lynx1.
... Previous work reported that AChR clusters can form spontaneously when myotubes are cultured on plates coated with laminin (Kummer et al., 2004). These AChR aggregates display several characteristic features of the mature postsynaptic apparatus, including colocalization of multiple postsynaptic proteins and clustering of subjacent myonuclei (Bruneau et al., 2005a(Bruneau et al., , 2005b. Accordingly, this culture system represents a relevant model to study postsynaptic membrane organization. ...
Article
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Microtubules (MTs) are known to be post-translationally modified at the neuromuscular junction (NMJ), hence increasing their stability. To date however, the function(s) of the dynamic MT network and its relative stability in the formation and maintenance of NMJs remain poorly described. Stabilization of the MT is dependent in part on its acetylation status, and HDAC6 is capable of reversing this post-translational modification. Here, we report that HDAC6 preferentially accumulates at NMJs and that it contributes to the organization and the stability of NMJs. Indeed, pharmacological inhibition of HDAC6 protects against MT disorganization and reduces the size of acetylcholine receptor (AChR) clusters. Moreover, the endogenous HDAC6 inhibitor paxillin interacts with HDAC6 in skeletal muscle cells, colocalizes with AChR aggregates, and regulates the formation of AChR. Our findings indicate that the focal insertion of AChRs into the postsynaptic membrane is regulated by stable MTs and highlight how an MT/HDAC6/paxillin axis participates in the regulation of AChR insertion and removal to control the structure of NMJs.
... This finding was later reproduced by several other studies using further, refined radiolabeling [154][155][156][157][158][159][160][161][162] or live-animal imaging methods with fluorescently labeled α-bungarotoxin in wildtype [163] and in transgenic nAChRγ-GFP mice [164]. The observations of nAChR endocytosis using electron microscopic analysis of electron-dense precipitates mediated by peroxidase-coupled α-bungarotoxin [165,166] and of a nAChR recycling pool by a smart α-bungarotoxin-biotin streptavidin-dye live imaging approach [167], paved the way for a series of further studies on the pathways leading to nAChR recycling and degradation. These used live imaging in combination with sequential application of differentially labeled α-bungarotoxins and often with heterologous expression of genetically encoded molecular biosensors. ...
Article
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By mediating voluntary muscle movement, vertebrate neuromuscular junctions (NMJ) play an extraordinarily important role in physiology. While the significance of the nerve-muscle connectivity was already conceived almost 2000 years back, the precise cell and molecular biology of the NMJ have been revealed in a series of fascinating research activities that started around 180 years ago and that continues. In all this time, NMJ research has led to fundamentally new concepts of cell biology, and has triggered groundbreaking advancements in technologies. This review tries to sketch major lines of thought and concepts on NMJ in their historical perspective, in particular with respect to anatomy, function, and molecular components. Furthermore, along these lines, it emphasizes the mutual benefit between science and technology, where one drives the other. Finally, we speculate on potential major future directions for studies on NMJ in these fields.
... Using a pulse-chase protocol to stain AChRs first with labeled α-bungaroxin, then following with unlabeled toxin after nerve addition, AChR aggregation under the nerve was shown to include receptors that were stained before nerve addition. These were interpreted as having migrated laterally through the muscle membrane [8], although endocytosis and re-insertion of labeled AChRs may also have contributed to their ultimate localization at the synapse [98]. This aggregation of AChRs was subsequently shown to proceed in parallel with a series of defined stages in the development of a specialized synaptic basal lamina [14,97]. ...
Article
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The vertebrate skeletal neuromuscular junction (NMJ) has long served as a model system for studying synapse structure, function, and development. Over the last several decades, a neuron-specific isoform of agrin, a heparan sulfate proteoglycan, has been identified as playing a central role in synapse formation at all vertebrate skeletal neuromuscular synapses. While agrin was initially postulated to be the inductive molecule that initiates synaptogenesis, this model has been modified in response to work showing that postsynaptic differentiation can develop in the absence of innervation, and that synapses can form in transgenic mice in which the agrin gene is ablated. In place of a unitary mechanism for neuromuscular synapse formation, studies in both mice and zebrafish have led to the proposal that two mechanisms mediate synaptogenesis, with some synapses being induced by nerve contact while others involve the incorporation of prepatterned postsynaptic structures. Moreover, the current model also proposes that agrin can serve two functions, to induce synaptogenesis and to stabilize new synapses, once these are formed. This review examines the evidence for these propositions, and concludes that it remains possible that a single molecular mechanism mediates synaptogenesis at all NMJs, and that agrin acts as a stabilizer, while its role as inducer is open to question. Moreover, if agrin does not act to initiate synaptogenesis, it follows that as yet uncharacterized molecular interactions are required to play this essential inductive role. Several alternatives to agrin for this function are suggested, including focal pericellular proteolysis and integrin signaling, but all require experimental validation.
... Using a pulse-chase protocol to stain AChRs first with labeled α-bungaroxin, then following with unlabeled toxin after nerve addition, AChR aggregation under the nerve was shown to include receptors that were stained before nerve addition. These were interpreted as having migrated laterally through the muscle membrane (Anderson and Cohen, 1977), although endocytosis and re-insertion of labeled AChRs may also have contributed to their ultimate localization at the synapse (Bruneau et al., 2005). This aggregation of AChRs was subsequently shown to proceed in parallel with a series of defined stages in the development of a specialized synaptic basal lamina Anderson, 1986). ...
Preprint
The vertebrate skeletal neuromuscular junction (NMJ) has long served as a model system for studying synapse structure, function and development. Over the last several decades a neuron-specific isoform of agrin, a heparan sulfate proteoglycan, has been identified as playing a central role in synapse formation at all vertebrate skeletal neuromuscular synapses. While agrin was initially postulated to be the inductive molecule that initiates synaptogenesis, this model has been modified in response to work showing that postsynaptic differentiation can develop in the absence of innervation, and that synapses can form in transgenic mice in which the agrin gene is ablated. In place of a unitary mechanism for neuromuscular synapse formation, studies in both mice and zebrafish have led to the proposal that two mechanisms mediate synaptogenesis, with some synapses being induced by nerve contact while others involve the incorporation of prepatterned postsynaptic structures. Moreover, the current model also proposes that agrin can serve two functions, to induce synaptogenesis and to stabilize new synapses, once these are formed. This review examines the evidence for these propositions, and concludes that it remains possible that a single molecular mechanism mediates synaptogenesis at all NMJs, and that agrin acts as a stabilizer, while its role as inducer is open to question. Moreover, if agrin does not act to initiate synaptogenesis, it follows that as yet uncharacterized molecular interactions are required to play this essential inductive role. Several alternatives to agrin for this function are suggested, including focal pericellular proteolysis and integrin signaling, but all require experimental validation.
... We show that the 3D co-culture platform, and not a 2D co-culture system, supports the transition from the embryonic to the adult AChR, thereby enabling the functional assessment of the adult neuromuscular junction in vitro. Our data align with prior studies showing that epsilon functional activity is regulated post-transcriptionally (Bruneau et al., 2005;Caroni et al., 1993;Jayawickreme and Claudio, 1994;Khan et al., 2014;Missias et al., 1996;Ross et al., 1991;Wild et al., 2016;Witzemann et al., 2013;Xu and Salpeter, 1997;Yampolsky et al., 2008), and in particular, supports work indicating a role for innervation (spontaneous miniature endplate potentials) and / or muscle fiber maturation in encouraging subunit substitution (Caroni et al., 6 disease by treating neuromuscular co-cultures with IgG purified from myasthenia gravis (MG) patient sera together with human complement, which results in readily visible clinical phenotypes in as early as two weeks of culture time. Thus, the described 3D co-culture model enables investigation of adult human NMJ development and therefore adult forms of neuromuscular diseases in vitro for the first time. ...
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Two-dimensional (2D) human skeletal muscle fiber cultures are ill equipped to support the contractile properties of maturing muscle fibers. This limits their application to the study of adult human neuromuscular junction (NMJ) development, a process requiring maturation of muscle fibers in the presence of motor neuron endplates. Here we describe a three-dimensional (3D) co-culture method whereby human muscle progenitors mixed with human pluripotent stem cell-derived motor neurons self-organize to form functional NMJ connections within two weeks. Functional connectivity between motor neuron endplates and muscle fibers is confirmed with calcium transient imaging and electrophysiological recordings. Notably, we only observed epsilon acetylcholine receptor subunit protein upregulation and activity in 3D co-culture. This demonstrates that the 3D co-culture system supports a developmental shift from the embryonic to adult form of the receptor that does not occur in 2D co-culture. Further, 3D co-culture treatments with myasthenia gravis patient sera shows the ease of studying human disease with the system. This work delivers a simple, reproducible, and adaptable method to model and evaluate adult human NMJ de novo development and disease in culture.
... The NMJ has been broadly used to understand the principles of synaptic organization and function 16,17 . Along with its relatively large size and easier accessibility in comparison to central nervous system synapses, the availability of fluorescent conjugates of α-bungarotoxin (BTX), which binds with high affinity to muscle nicotinic acetylcholine receptors (nAChRs) 18 , have facilitated a deep understanding of postsynaptic organization at the NMJ, including the distribution, stability, and trafficking of nAChRs [19][20][21][22][23][24] . ...
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Lithium chloride has been widely used as a therapeutic mood stabilizer. Although cumulative evidence suggests that lithium plays modulatory effects on postsynaptic receptors, the underlying mechanism by which lithium regulates synaptic transmission has not been fully elucidated. In this work, by using the advantageous neuromuscular synapse, we evaluated the effect of lithium on the stability of postsynaptic nicotinic acetylcholine receptors (nAChRs) in vivo. We found that in normally innervated neuromuscular synapses, lithium chloride significantly decreased the turnover of nAChRs by reducing their internalization. A similar response was observed in CHO-K1/A5 cells expressing the adult muscle-type nAChRs. Strikingly, in denervated neuromuscular synapses, lithium led to enhanced nAChR turnover and density by increasing the incorporation of new nAChRs. Lithium also potentiated the formation of unstable nAChR clusters in non-synaptic regions of denervated muscle fibres. We found that denervation-dependent re-expression of the foetal nAChR γ-subunit was not altered by lithium. However, while denervation inhibits the distribution of β-catenin within endplates, lithium-treated fibres retain β-catenin staining in specific foci of the synaptic region. Collectively, our data reveal that lithium treatment differentially affects the stability of postsynaptic receptors in normal and denervated neuromuscular synapses in vivo, thus providing novel insights into the regulatory effects of lithium on synaptic organization and extending its potential therapeutic use in conditions affecting the peripheral nervous system.
... The area on the postsynaptic membrane of AChR density grows, and AChRs are added as muscle fibers grow in size (Balice-Gordon & Lichtman, 1990). AChRs are also recycled (Bruneau, Sutter, Hume, & Akaaboune, 2005), and junctional folds form and increase in number (Desaki & Uehara, 1987). In an investigation of morphological changes in the human NMJ across four age groups (children, young adults, old adults, and aged), Wokke et al. (1990) described decreased size of nerve terminals between the child and young adult groups and increased length and branching of the postsynaptic membrane throughout life. ...
Article
Purpose The aim of the current study was to review neuromuscular development, summarize the current body of evidence describing the use of neuromuscular electrical stimulation (NMES) therapy in infants, and identify possible contraindications for the use of NMES in the neonate and young infant. Method After a review of the literature describing neuromuscular development, we created a timeline of the developmental processes. Key milestones were determined, and a literature search was conducted to identify potential effects of electrical stimulation on this process. Results Current evidence supporting the use of NMES in the pediatric population is limited and of poor quality. Contraindications of the use of NMES in the neonate and young infant were identified, including (a) inhibited expression of the neural cell adhesion molecule that is vital for neuromuscular development, (b) alteration of muscle fiber type metabolic profile away from intended muscle fiber type morphology, and (c) interruption of postsynaptic acetylcholine receptor synthesis during neuromuscular junction development. Conclusion The use of NMES for the treatment of dysphagia in the neonate and young infant may influence early neuromuscular development in a manner that is not currently well understood. Future research is needed to further understand the effects of NMES on the developing neuromuscular system.
... We show that the 3D co-culture platform, and not a 2D co-culture system, supports the transition from the embryonic to the adult AChR, thereby enabling the functional assessment of the adult neuromuscular junction in vitro. We present data aligning with prior studies showing that epsilon functional activity is regulated posttranscriptionally (Bruneau et al., 2005;Caroni et al., 1993;Jayawickreme and Claudio, 1994;Khan et al., 2014;Missias et al., 1996;Ross et al., 1991;Wild et al., 2016;Witzemann et al., 2013;Xu and Salpeter, 1997;Yampolsky et al., 2008), and in particular, supports work indicating a role for innervation (spontaneous miniature endplate potentials) and/or muscle fiber maturation in encouraging subunit substitution (Caroni et al., 1993;Missias et al., 1996;Witzemann et al., 2013;Xu and Salpeter, 1997;Yampolsky et al., 2008). Finally, we demonstrate the versatility and ease of the system for modeling human disease by treating neuromuscular co-cultures with IgG purified from myasthenia gravis (MG) patient sera together with human complement, which results in readily visible clinical-like phenotypes in as early as two weeks of culture time. ...
Article
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Two-dimensional (2D) human skeletal muscle fiber cultures are ill-equipped to support the contractile properties of maturing muscle fibers. This limits their application to the study of adult human neuromuscular junction (NMJ) development, a process requiring maturation of muscle fibers in the presence of motor neuron endplates. Here we describe a three-dimensional (3D) co-culture method whereby human muscle progenitors mixed with human pluripotent stem cell-derived motor neurons self-organize to form functional NMJ connections. Functional connectivity between motor neuron endplates and muscle fibers is confirmed with calcium imaging and electrophysiological recordings. Notably, we only observed epsilon acetylcholine receptor subunit protein upregulation and activity in 3D co-cultures. Further, 3D co-culture treatments with myasthenia gravis patient sera shows the ease of studying human disease with the system. Hence, this work offers a simple method to model and evaluate adult human NMJ de novo development or disease in culture.
... We tried to label cryptococcal cells with EZ-Link NHS-biotin and Oregon green 488-conjugated NeutrAvidin, and phagocytosis was performed using guinea pig complement as an opsonin. However, the signal of Oregon green was not stable in the phagosome, and after 24 h, it was lost completely, which we attribute to dye degradation from a combination of the low phagosomal pH and the oxidative burst (20). We have also tried to measure the phagolysosomal pH with heat-killed cryptococcal cells labeled with NHS-biotin, but the labeling did not work with heat-killed cells. ...
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Cryptococcus neoformans is the causative agent of cryptococcosis, a devastating fungal disease that affects thousands of individuals worldwide. This fungus has the capacity to survive inside phagocytic cells, which contributes to persistence of infection and dissemination. One of the major antimicrobial mechanisms of host phagocytes is to acidify the phagosomal compartment after ingestion of microbes. This study shows that the capsule of C. neoformans can interfere with full phagosomal acidification by serving as a buffer.
... Synaptic AChRs are internalized and traffic back to the membrane "recycled." This recycling is dependent upon synaptic activity [113]. Notably, such recycled AChRs have a shorter half-life than nonrecycled receptors [114]. ...
Article
The neuromuscular junction (NMJ) is the vehicle for fast, reliable and robust communication between motor neuron and muscle. The unparalleled accessibility of this synapse to morphological, electrophysiological and genetic analysis has yielded an in depth understanding of many molecular components mediating its formation, maturation and stability. However, key questions surrounding the signaling pathways mediating these events and how they play out across the lifetime of the synapse remain unanswered. Such information is critical since the NMJ is necessary for normal movement and is compromised in several settings including Myasthenia Gravis, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), muscular dystrophy, sarcopenia and aging. Muscle specific kinase (MuSK) is a central player in most if not all contexts of NMJ formation and stability. However, elucidating the function of this receptor in this range of settings is challenging since MuSK participates in at least three signaling pathways: as a tyrosine kinase-dependent receptor for agrin-LRP4 and Wnts; and, as a kinase-independent BMP co-receptor. Here we focus on NMJ stability during aging and discuss open questions regarding the molecular mechanisms that govern active maintenance of the NMJ, with emphasis on MuSK and the potential role of its multiple signaling contexts.
... It has been shown previously that the endplate AChR clusters (i.e. AChR clusters at the postsynaptic muscle membrane of the neuromuscular junction) are relatively stable and have a long half-life, but recycling and intra-junctional migration of receptors are important processes that contribute to cluster dynamics (Akaaboune et al., 1999(Akaaboune et al., , 2002Bruneau et al., 2005). In support with this notion, we found that active Rac1 rarely colocalizes with high-density AChR clusters; instead it is mainly present in close proximity to the clusters. ...
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Rac1, an important Rho GTPase that regulates actin cytoskeleton, has long been suggested to participate in acetylcholine receptor (AChR) clustering at the postsynaptic neuromuscular junction. However, how Rac1 is regulated and how it influences AChR clusters have remained unexplored. This study shows that breaking the balance of Rac1 regulation, by either increasing or decreasing its activity, led to impaired formation and maintenance of AChR clusters. By manipulating Rac1 activity at different stages of AChR clustering in cultured myotubes, we showed that Rac1 activation was required for the initial formation of AChR clusters, but its persistent activation led to AChR destabilization, and uncontrolled hyperactivation of Rac1 even caused excessive myotube fusion. Both AChR dispersal and myotube fusion induced by Rac1 were dependent on its downstream effector Pak1 (p21-activating kinase 1). Two Rac1 GAPs and Six Rac1 GEFs were screened to be important for normal AChR clustering. Together, this study reveals that whereas general Rac1 activity remains at low levels during terminal differentiation of myotubes and AChR cluster maintenance, tightly regulated Rac1 activity controls normal AChR clustering.
... During early postnatal life, the fetal form of the AChR, containing a gamma subunit (2α:ß:δ:γ) is gradually replaced by an epsilon subunit-containing adult form (2α:ß:δ:ε), leading to increased calcium conductance of the receptor (Missias et al., 1996). The half-life of synaptic AChRs also increases during maturation as insertion of new AChRs and the recycling of internalized AChRs maintain the high density of AChRs at the crests of postsynaptic junctional folds (Bruneau et al., 2005;Bruneau and Akaaboune, 2006). ...
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The neuromuscular junction (NMJ) is a highly specialized synapse between a motor neuron nerve terminal and its muscle fiber that are responsible for converting electrical impulses generated by the motor neuron into electrical activity in the muscle fibers. On arrival of the motor nerve action potential, calcium enters the presynaptic terminal, which leads to the release of the neurotransmitter acetylcholine (ACh). ACh crosses the synaptic gap and binds to ACh receptors (AChRs) tightly clustered on the surface of the muscle fiber; this leads to the endplate potential which initiates the muscle action potential that results in muscle contraction. This is a simplified version of the events in neuromuscular transmission that take place within milliseconds, and are dependent on a tiny but highly structured NMJ. Much of this review is devoted to describing in more detail the development, maturation, maintenance and regeneration of the NMJ, but first we describe briefly the most important molecules involved and the conditions that affect their numbers and function. Most important clinically worldwide, are myasthenia gravis (MG), the Lambert-Eaton myasthenic syndrome (LEMS) and congenital myasthenic syndromes (CMS), each of which causes specific molecular defects. In addition, we mention the neurotoxins from bacteria, snakes and many other species that interfere with neuromuscular transmission and cause potentially fatal diseases, but have also provided useful probes for investigating neuromuscular transmission. There are also changes in NMJ structure and function in motor neuron disease, spinal muscle atrophy and sarcopenia that are likely to be secondary but might provide treatment targets. The NMJ is one of the best studied and most disease-prone synapses in the nervous system and it is amenable to in vivo and ex vivo investigation and to systemic therapies that can help restore normal function.
... On day 19 of neural differentiation, dissociated UC005-MNs were added to muscle culture cell dishes, and after 5 days of coculturing, muscle cell contraction was observed with a brightfield microscope. Within additional 2-3 days, immunofluorescence staining revealed positive labeling for the acetylcholine receptor (AChR) marker, α-bungarotoxin (α-BTX), on the surface of the muscle cells [21] and colocalization of α-BTX and TuJ1 at sites on the axons of the UC005-MNs (Figure 6(b)), which indicated accumulation of AChR and the formation of NMJs between urine-MNs and muscle cells at these sites [22]. ...
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Induced pluripotent stem cells (iPSCs) have provided new opportunities for motor neuron disease (MND) modeling, drug screening, and cellular therapeutic development. Among the various types of iPSCs, urine-derived iPSCs have become a promising source of stem cells because they can be safely and noninvasively isolated and easily reprogrammed. Here, for the first time, we differentiated urine-derived iPSCs (urine-iPSCs) into motor neurons (MNs) and compared the capacity of urine-iPSCs and cord-blood-derived iPSCs (B-iPSCs) to differentiate into MNs. With the use of small molecules, mature MNs were generated from urine-iPSCs as early as 26 days in culture. Furthermore, in coculture with muscle cells, MNs projected long axons and formed neuromuscular junctions (NMJs). Immunofluorescence and PCR confirmed the expression levels of both MN and NMJ markers. The comparison of the ratios of positive labeling for MN markers between urine-iPSCs and B-iPSCs demonstrated that the differentiation potentials of these cells were not significantly different. The abovementioned results indicate that urine-iPSCs are a new, promising source of stem cells for MND modeling and further cellular therapeutic development.
... Briefly, 6-hydroxy dopamine (6OHD) was injected into mouse hindlimb tibialis anterior for 2 weeks every other day. Similar to previous reports (Akaaboune et al., 1999;Bruneau et al., 2005;Röder et al., 2009Röder et al., , 2010Choi et al., 2012;Khan et al., 2014Khan et al., , 2016, stability of AChR was addressed using a sequential labeling approach, where old and newly formed pools of AChR were marked with BGT 647 and BGT 555, respectively. Subsequent in vivo confocal analysis showed that upon sympathectomy, the relative amount of BGT 555 staining increased over that of BGT 647 (Figure 2A). ...
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Recent studies have demonstrated that neuromuscular junctions are co-innervated by sympathetic neurons. This co-innervation has been shown to be crucial for neuromuscular junction morphology and functional maintenance. To improve our understanding of how sympathetic innervation affects nerve–muscle synapse homeostasis, we here used in vivo imaging, proteomic, biochemical, and microscopic approaches to compare normal and sympathectomized mouse hindlimb muscles. Live confocal microscopy revealed reduced fiber diameters, enhanced acetylcholine receptor turnover, and increased amounts of endo/lysosomal acetylcholine-receptor-bearing vesicles. Proteomics analysis of sympathectomized skeletal muscles showed that besides massive changes in mitochondrial, sarcomeric, and ribosomal proteins, the relative abundance of vesicular trafficking markers was affected by sympathectomy. Immunofluorescence and Western blot approaches corroborated these findings and, in addition, suggested local upregulation and enrichment of endo/lysosomal progression and autophagy markers, Rab 7 and p62, at the sarcomeric regions of muscle fibers and neuromuscular junctions. In summary, these data give novel insights into the relevance of sympathetic innervation for the homeostasis of muscle and neuromuscular junctions. They are consistent with an upregulation of endocytic and autophagic trafficking at the whole muscle level and at the neuromuscular junction.
... Enfin, le marquage par une protéine fluorescente sensible au pH, telle que la pHluorine, permet de distinguer les récepteurs synaptiques des récepteurs subsynaptiques (alternativement, une injection in vivo d'anticorps dans la cavité pseudocoelomique permet d'obtenir la même information). (Bruneau et al., 2005). Ces mécanismes d'endocytose et recyclage constants sont modulés par l'activité, et permettent de contrôler le nombre de récepteurs, et donc la force de la synapse. ...
Thesis
Au cours du développement du système nerveux, l'activité des cibles post-synaptiques permet le raffinement du nombre et de la force des connexions neuronales. En employant la jonction neuromusculaire de Caenorhabditis elegans comme système modèle, nous avons étudié deux aspects de la mise en place de ces connexions. D'une part, nous montrons que le nombre de récepteurs présents à la jonction neuromusculaire est contrôlé par l'activité musculaire : une augmentation de l'activation synaptique entraîne une régulation différentielle des trois types de récepteurs présents à la jonction neuromusculaire. D'autre part, nous avons étudié les changements de la morphologie de certains motoneurones de la tête du ver, appelés neurones SAB, en fonction de l’activité musculaire. Une diminution de l’activité musculaire durant une période critique du développement entraîne une surcroissance axonale des neurones SAB. À travers différentes approches, nous avons pu identifier la suppression de la surcroissance axonale dans des mutants où la biosynthèse des neuropeptides est perturbée. Enfin, nous avons mis en évidence que la surcroissance axonale apparait également lors de perturbations plus générales de la physiologie cellulaire, telles qu'un choc thermique ou la surexpression d'un transgène, ce qui suggère que le système SAB est plastique et particulièrement sensible au cours du développement
... In normal innervated muscle fibers, the AChRs are highly concentrated at the crests of the junctional folds, where they are held in clusters to ensure synaptic transmission. This stability of AChRs is established through an equilibrium between rates of removal and insertion of AChRs (Bruneau et al., 2005;Bruneau and Akaaboune, 2006;Martinez-Pena y Valenzuela et al., 2011). At functioning NMJs, receptor lifetime in the junctional membrane is quite long; however, the lifetime of DGC proteins at NMJs remains unknown. ...
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Bimolecular fluorescence complementation (BiFC) approach was used to study the molecular interactions between different components of the postsynaptic protein complex at the neuromuscular junction of living mice. Here, we showed that rapsyn forms complex with both ?-dystrobrevin and ?-syntrophin at the crests of junctional folds. The linkage of rapsyn to ?-syntrophin and/or ?-dystrobrevin is mediated by utrophin, a protein localized at AChR-rich domain. In mice deficient in ?-syntrophin, in which utrophin is no longer present at the synapse, rapsyn interaction with ?- dystrobrevin was completely abolished. This interaction was completely restored when either utrophin or ?-syntrophin was introduced into muscles deficient in ?-syntrophin. However, in NMJ deficient in ?-dystrobrevin, in which utrophin remains intact, complex formation between rapsyn and ?-syntrophin was not affected. Using fluorescence recovery after photobleaching, we found that ?-syntrophin is turning over 5-7 times faster than AChRs and loss of ?-dystrobrevin has no effect on rapsyn and ?-syntrophin half-lives whereas the half-life of AChR is significantly altered. Altogether, these results provide new insights into the spatial distribution of dystrophin glycoprotein components and their dynamisms in living mice.
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The strong interaction between streptavidin (SA) and biotin is widely utilized in biotechnological applications. A SA variant, monovalent SA, was developed with a single and high affinity biotin-binding site within the intact tetramer. However, its structural characterization remains undetermined. Here, we seek to determine the crystal structure of monovalent SA at 1.7-Å resolution. We show that, in contrast to its ‘close-state’ in the only wild-type subunit, the L3,4 loops of three Dead SA subunits are free from crystal packing and remain in an ‘open state’, stabilized by a consistent H-bonding network involving S52. This H-bonding network also applies to the previously reported open state of the wild-type apo-SA. These results suggest that specific substitutions (N23A/S27D/S45A) at biotin-binding sites stabilize the open state of SA L3,4 loop, thereby further reducing biotin-binding affinity. The general features of the ‘open state’ SA among different SA variants may facilitate its rational design. The structural information of monovalent SA will be valuable for its applications across a wide range of biotechnological areas.
Article
At the neuromuscular junction (NMJ), the postsynaptic localization of muscle acetylcholine receptor (AChR) is regulated by neural signals and occurs via several processes including metabolic stabilization of the receptor. However, the molecular mechanisms that influence receptor stability remain poorly defined. Here, we show that neural agrin and the tyrosine phosphatase inhibitor, pervanadate slow the degradation of surface receptor in cultured muscle cells. Their action is mediated by tyrosine phosphorylation of the AChR β subunit, as agrin and pervandate had no effect on receptor half‐life in AChR‐β3F/3F muscle cells, which have targeted mutations of the β subunit cytoplasmic tyrosines. Moreover, in wild type AChR‐β3Y muscle cells, we found a linear relationship between average receptor half‐life and the percentage of AChR with phosphorylated β subunit, with half‐lives of 12.7 and 23 h for nonphosphorylated and phosphorylated receptor, respectively. Surprisingly, pervanadate increased receptor half‐life in AChR‐β3Y myotubes in the absence of clustering, and agrin failed to increase receptor half‐life in AChR‐β3F/3F myotubes even in the presence of clustering. The metabolic stabilization of the AChR was mediated specifically by phosphorylation of βY390 as mutation of this residue abolished β subunit phosphorylation but did not affect δ subunit phosphorylation. Receptor stabilization also led to higher receptor levels, as agrin increased surface AChR by 30% in AChR‐β3Y but not AChR‐β3F/3F myotubes. Together, these findings identify an unexpected role for agrin‐induced phosphorylation of βY390 in downregulating AChR turnover. This likely stabilizes AChR at developing synapses, and contributes to the extended half‐life of AChR at adult NMJs. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 399–410, 2013
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A muscle-specific nonkinase anchoring protein (αkap), encoded within the calcium/calmodulin kinase II (camk2) α gene, was recently found to control the stability of acetylcholine receptor (AChR) clusters on the surface of cultured myotubes. However, it remains unknown whether this protein has any effect on receptor stability and the maintenance of the structural integrity of neuromuscular synapses in vivo. By knocking down the endogenous expression of αkap in mouse sternomastoid muscles with shRNA, we found that the postsynaptic receptor density was dramatically reduced, the turnover rate of receptors at synaptic sites was significantly increased, and the insertion rates of both newly synthesized and recycled receptors into the postsynaptic membrane were depressed. Moreover, we found that αkap shRNA knockdown impaired synaptic structure as postsynaptic AChR clusters and their associated postsynaptic scaffold proteins within the neuromuscular junction were completely eliminated. These results provide new mechanistic insight into the role of αkap in regulating the stability of the postsynaptic apparatus of neuromuscular synapses. Copyright © 2015 the authors 0270-6474/15/355118-10$15.00/0.
Chapter
The neuromuscular junction (NMJ) is a cholinergic synapse that connects a motor neuron to a skeletal muscle fiber. To enable sustained tetanic contraction of skeletal muscle, the NMJ must reliably transmit the impulses from the presynaptic motor neuron to the postsynaptic muscle fiber. This seemingly simple task is enabled by the existence of a complex system of pre- and postsynaptic structural subcellular specializations and functional molecular machineries. These are responsible for (1) the development and maintenance of the synaptic structure, (2) the controlled presynaptic release of the neurotransmitter acetylcholine, and (3) the postsynaptic translation of this chemical message into an excitatory electrical response. The many factors in this synaptic system all have their inherent risks and vulnerabilities, e.g., in autoimmunity or upon intoxication. The resulting malfunctions compromise successful neuromuscular transmission and may lead to disturbances of muscle contraction. This chapter describes the normal NMJ structure and electrophysiological function and briefly discusses the pathophysiology occurring in myasthenia gravis (with autoantibodies against postsynaptic acetylcholine receptors) and some of the related NMJ synaptopathies.
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Chemical modification of proteins provides great opportunities to control and visualize living systems. The most common way to modify proteins is reaction of their abundant amines with N-hydroxysuccini-mide (NHS) esters. Here we explore the impact of amine number and positioning on protein-conjugate behavior using streptavidin-biotin, a central research tool. Dye-NHS modification of streptavidin severely damaged ligand binding, necessitating development of a new streptavidin-retaining ultrastable binding after labeling. Exploring the ideal level of dye modification , we engineered a panel bearing 1–6 amines per subunit: ''amine landscaping.'' Surprisingly, brightness increased as amine number decreased, revealing extensive quenching following conventional labeling. We ultimately selected Flavidin (fluo-rophore-friendly streptavidin), combining ultrastable ligand binding with increased brightness after conjugation. Flavidin enhanced fluorescent imaging, allowing more sensitive and specific cell labeling in tissues. Flavidin should have wide application in molecular detection, providing a general insight into how to optimize simultaneously the behavior of the biomolecule and the chemical probe.
Article
α-syntrophin (α-syn) and α-dystrobrevin (α-dbn), two components of the dystrophin-glycoprotein complex, are essential for the maturation and maintenance of the neuromuscular junction (NMJ) and mice deficient in either α-syn or α-dbn exhibit similar synaptic defects. However, the functional link between these two proteins and whether they exert distinct or redundant functions in the postsynaptic organization of the NMJ remain largely unknown. We generated and analyzed the synaptic phenotype of double heterozygote (α-dbn+/−, α-syn+/−), and double homozygote knockout (α-dbn−/−; α-syn−/−) mice and examined the ability of individual molecules to restore their defects in the synaptic phenotype. We showed that in double heterozygote mice, NMJs have normal synaptic phenotypes and no signs of muscular dystrophy. However, in double knockout mice (α-dbn−/−; α-syn−/−), the synaptic phenotype (the density, the turnover, and the distribution of AChRs within synaptic branches) is more severely impaired than in single α-dbn−/− or α-syn−/− mutants. Furthermore, double mutant and single α-dbn−/− mutant mice showed more severe exercise-induced fatigue and more significant reductions in grip strength than single α-syn−/− mutant and wild-type. Finally, we showed that the overexpression of the transgene α-syn-GFP in muscles of double mutant restores primarily the abnormal extensions of membrane containing AChRs that extend beyond synaptic gutters and lack synaptic folds, whereas the overexpression of α-dbn essentially restores the abnormal dispersion of patchy AChR aggregates in the crests of synaptic folds. Altogether, these data suggest that α-syn and α-dbn act in parallel pathways and exert distinct functions on the postsynaptic structural organization of NMJs.
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In order to study how neuromuscular junctions grow, we have repeatedly viewed the same junctions in mouse sternomastoid muscles at monthly intervals from 2 weeks to 18 months of age. Motor nerve terminals were stained with the nontoxic fluorescent dye 4-Di-2-ASP (Magrassi et al., 1987), and postsynaptic ACh receptors were labeled with fluorescently tagged alpha-bungarotoxin. Neuromuscular junctions grew primarily by expansion of existing motor nerve terminal and postsynaptic receptor regions without the addition or loss of synaptic areas. The expansion of pre- and postsynaptic specializations was precisely matched, suggesting that as neuromuscular junctions grow, the opposing specializations enlarge simultaneously. Each neuromuscular junction grew in length and width at the same rate that muscle fibers enlarged in those 2 dimensions, suggesting that junctional growth might be a mechanical consequence of muscle fiber growth. Repeated visualization of ACh receptors over time showed that previously labeled receptors spread apart in the membrane occupying a progressively larger area as muscle fibers grew. At the same time, new receptors were intercalated throughout the enlarged postsynaptic area. Thus, the growth of postsynaptic regions appears to be directly related to the expansion of the muscle fiber membrane as muscle fibers grow. The maintained alignment between growing motor nerve terminals and postsynaptic regions suggests that nerve terminal growth may be a consequence of its adhesion to growing postsynaptic specializations. This conclusion is supported by the coextensive stretching of motor nerve terminals and postsynaptic regions when muscle fibers are stretched. Thus, the growth of motor nerve terminals is coupled to the growth of postsynaptic regions, and the growth of the postsynaptic regions is in turn coupled to the growth of muscle fibers. In this way, the branching pattern of neuromuscular junctions may be stably maintained despite ongoing enlargement of synaptic area.
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Identified neuromuscular junctions were followed in the sternomastoid muscle of living mice for several months by repeated staining with the fluorescent dye 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-ASP; Magrassi et al., 1987). Overall terminal growth occurred at many endplates; however, the branching pattern of presynaptic arbors was largely unchanged, even after several months. The absence of significant remodeling over time was not a result of dye-staining, since sprouting was readily induced at residual motor endings by partial denervation. We conclude that--apart from overall growth--most neuromuscular junctions in the adult mouse are stable over intervals that represent a significant fraction of the animal's lifetime.
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The principal aim of this work was to better understand how regenerating muscle fibers become innervated in adult animals. To induce muscle regeneration, individual identified muscle fibers in a mouse were damaged with a laser focused through a microscope. The muscle fiber that degenerated and the muscle fiber that was formed in its place were followed by viewing the same site repeatedly over a period of 2 d to 40 weeks. Commonly, the nerve terminal innervating the irradiated muscle fiber partially retracted during muscle fiber degeneration, and then sprouted to innervate the regenerating muscle fiber at the same site it had previously innervated the muscle fiber that was damaged. During the early phase of muscle regeneration we also observed sprouts originating from nerve terminals on adjacent muscle fibers. The new nerve growth was a response to the regenerating muscle fiber rather than to the degenerated fiber it replaced because repeated damage of the same site every 2-3 d over a 10 d period (to prevent regeneration) did not cause any sprouting. The direction of the sprouts on adjacent muscle fibers showed a bias toward the regenerating muscle fiber, although they avoided the region occupied by the original nerve terminal. Forty percent of the sprouts managed to reach the regenerated fiber. Nonetheless, by 11 d after muscle fiber damage, all sprouts had regressed, leaving the new fiber innervated by the same motor axon that innervated the fiber that was damaged. On the other hand, when the overlying nerve terminal as well as the muscle fiber was damaged, the sprouts from nearby muscle fibers were both more numerous and more stable, and in five cases we observed two or more new synaptic junctions on the regenerating fiber originating from different axons. In one case we witnessed a protracted competition between the original motor axon as it sprouted back and the sprouts from nearby junctions for sole innervation of the regenerate. Ultimately, the surviving sprouts myelinated and became the permanent and exclusive input to the new fiber. These results indicate that regenerating muscle fibers emit a signal that induces directional sprouting from nearby undamaged nerve terminals. Reinnervation of the regenerating muscle fiber by one axon apparently prevents the maintenance of such neurites. Because the process of muscle regeneration shares many features in common with myogenesis during embryonic development, it is likely that developing muscle fibers present an analogous stimulus to ingrowing motor axons.(ABSTRACT TRUNCATED AT 400 WORDS)
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The effect of electrical stimulation on the stability of junctional ACh receptors (AChR) on soleus muscles of Wistar rats was compared to that of denervation and reinnervation. Denervation causes the degradation rate of the slowly degrading AChRs (Rs) at the neuromuscular junction to accelerate and be replaced by rapidly degrading AChRs (Rr), while reinnervation restabilizes the accelerated Rs. Electrical stimulation initiated at the time of denervation prevented the acceleration of the Rs. It could not, however, reverse the effect of denervation if initiated after the AChRs became destabilized, nor could it slow the degradation rate of the Rr. We conclude that electrical stimulation of denervated muscle downregulates the expression of the Rr and prevents the destabilization of Rs.
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Activity-dependent changes in synaptic function are believed to underlie the formation of memories. A prominent example is long-term potentiation (LTP), whose mechanisms have been the subject of considerable scrutiny over the past few decades. I review studies from our laboratory that support a critical role for AMPA receptor trafficking in LTP and experience-dependent plasticity.
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Long-term potentiation (LTP) of synaptic strength, the most established cellular model of information storage in the brain, is expressed by an increase in the number of postsynaptic AMPA receptors. However, the source of AMPA receptors mobilized during LTP is unknown. We report that AMPA receptors are transported from recycling endosomes to the plasma membrane for LTP. Stimuli that triggered LTP promoted not only AMPA receptor insertion but also generalized recycling of cargo and membrane from endocytic compartments. Thus, recycling endosomes supply AMPA receptors for LTP and provide a mechanistic link between synaptic potentiation and membrane remodeling during synapse modification.
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We have developed a technique to measure changes in the amount of fluorescently labeled acetylcholine receptors in living muscles over long time periods. The measurements of fluorescence are made relative to a novel, photolytically stable fluorescence standard (Spectralon) which allows changes in fluorescence to be followed over days, even months. The method compensates for spatial and temporal variations in image brightness due to the light source, microscope, and camera. We use this approach to study the turnover of fluorescently labeled acetylcholine receptors at a single neuromuscular junction in a living mouse by re-imaging the same junction in situ over a period of 3 weeks. In addition we show that the SIT video camera, which is generally considered inadequate for quantitative imaging (in comparison to CCD cameras), is actually a very good quantitative device, especially in situations requiring both fast acquisition and high resolution.
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[125I mono-iodo-alpha-bungarotoxin is used as a specific marker in a description of acetylcholine receptor metabolism. It is concluded that acetylcholine receptors in the surface membranes of chick and rat myotubes developing in cell cultures have a half-life of 22-24 h. Alpha-bungarotoxin (bound to a receptor which is removed from the membrane) is degraded to monoiodotyrosine which appears in the medium. Several observations are consistent with a model in which receptors or alpha-bungarotoxin-receptor complexes are internalized and then degraded: (a) the rate of appearance of iodotyrosine does not reach its maximal rate until 90 min after alpha-bungarotoxin is bound to the surface receptors; (b) 2,4-dinitrophenol, reduced temperature, and cell disruption all inhibit the degradation process. The degradation of surface receptors is not coupled to the process by which receptors are incorporated into the membrane. Evidence suggest that receptors are incorporated into the surface membrane from a presynthesized set of receptors containing about 10% as many alpha-bungarotoxin binding sites as does the surface. Additionally, a third set of acetylcholine receptors is described containing about 30% as amny binding sites as does the surface. These "hidden" recptors are not precursors yet are not readily accessible for binding of extracellular alpha-bungarotoxin. These findings are discussed in relation to both plasma membrane biosynthesis and control of chemosensitivity in developing and denervated skeletal muscle.
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Alpha-Bungarotoxin is one of a class of proteins, isolated from snake venoms, which antagonize the action of acetylcholine at vertebrate neuromuscular junctions and 'electroplaques' of electric fish. Alpha-Bungarotoxin blocks acetylcholine action irreversibly and may be labelled with either 125I or 3H. This irreversible binding is used as the basis of an in vitro assay for acetylcholine receptors, whether in intact tissue, membrane fragments or solubilized preparations. Acetylcholine receptors from Torpedo and denervated skeletal muscle have been solutilized and substantially purified using affinity chromatography. The distribution of acetylcholine receptors in several tissues has been determined, and an auto-immune response, induced by injection of purified Torpedo receptors, has been studied.
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This review covers the literature related to the chemical structure of the receptor (as far as it is relevant to receptor regulation), the biosynthesis and incorporation of receptors into plasma membranes, the organization of receptors in postsynaptic membrane and extrasynaptic membrane, the degradation of receptors, and the manner in which receptor metabolism and organization are regulated during myogenesis and the formation of neuromuscular junctions, during adult life and in denervation and reinnervation of adult muscle fibers.
Tremorine pretreatment of mice induces tolerance to some effects of oxotremorine. In the state of tolerance even the highest doses of oxotremorine did not cause antinociception, this blockade being insurmountable. Oxotremorine decreased motility and amphetamine hypermotility, and both effects were diminished by tremorine pretreatment. Amphetamine hypermotility increased in the tolerance state. The increase of cerebral acetylcholine level due to oxotremorine was diminished by tremorine pretreatment. It is suggested that a special blockade of cerebral muscarinic receptors might play a role in the tolerance phenomenon, moreover it is possible that some excitation develops in the CNS. A homeostatic adaptation may be involved a role in this kind of tolerance.
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Multiple factors alter the interaction of muscle relaxants with the NMJ. This review has focused on the aberrant responses caused principally by alterations in AChRs (table 1). Many pathologic states increase (up-regulate) AChR number. These include upper and lower motor neuron lesions, muscle trauma, burns, and immobilization. Pre- or postjunctional inhibition of neurotransmission by drugs or toxins also up-regulate AChRs. These include alpha- and beta-BT, NDMR, anticonvulsants, and clostridial toxins. We speculate that other bacterial toxins also up-regulate AChR. With proliferation of AChRs, agonist drug dose-response curves are shifted to the left. The exaggerated release of potassium when depolarization occurs with the use of agonists such as SCh and decamethonium can be attributed to the increased number of AChR. Thus, SCh should be avoided in patients who are in the susceptible phase (see section V). In the presence of increased AChR, the requirement for NDMR is markedly increased. Thus, the response to NDMR may be used as an indirect estimator of increased sensitivity to SCh (table 1). The most extensively studied pathologic state in which there is a decrease in AChRs is myasthenia gravis; there is immunologically mediated destruction and/or functional blockade of AChRs. The pathophysiologic and pharmacologic changes in LEMS are quite distinct from those of myasthenia gravis. Decreased AChRs in myasthenia gravis result in resistance to agonists and increased sensitivity to competitive antagonists. In conditioning exercise, the perturbed muscles show sensitivity to NDMR that may be due to decreased AChRs. Chronic elevations of ACh observed with organophosphorus poisoning or chronic use of reversible cholinesterase inhibitors results in down-regulation of AChRs. In this condition, SCh should be avoided because its metabolic breakdown would be impaired; the requirement for NDMR may be decreased. All of the varied responses to SCh and NDMR, which are associated with concomitant changes in AChRs, are analogous to drug-receptor interactions observed in other biologic systems.
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Clearly, many aspects of the action of neuromuscular blockers remain poorly understood at the molecular level. In the case of competitive blockers, blockade of EPPs by competitive binding to the ACh receptor site accounts for the most clinically important aspect of blockade. Although train-of-four fade produced by curare and some other competitive agents probably represents a presynaptic action, the molecular mechanisms underlying this effect have not been addressed. Depolarizing blockade is inherently more complicated than competitive blockade. Simple depolarization and inactivation of the mechanism for generation of the action potential probably account for the major clinical effect seen in phase I block. Furthermore, the relative balance between activation of channels and desensitization may also provide a qualitative explanation for phase II block and tachyphylaxis. However, effects that are more likely to be explained by presynaptic actions of depolarizing blockers have also been described, and it is not yet possible to assess quantitatively whether the rates of onset of the different postsynaptic actions can account for most aspects of depolarizing block. This discussion has raised several issues which need to be addressed in future studies. 1. What are the presynaptic effects of cholinergic drugs? Do these drugs act through a specific receptor or, on other ion channels in the terminal membrane, or do they operate by mechanisms distinct from effects on membrane conductance? Can any of the observations be explained by indirect effects mediated through postsynaptic ACh receptors, e.g., K+ release? 2. What are the factors that influence variability in sensitivity to neuromuscular blockers among species, muscles within species, and during development? Many of the potential factors, e.g., differences in safety factor, resting conductances, ACh receptor type, and differences in the presence and absence of presynaptic receptors, have been outlined, but definitive tests of the contribution of any particular mechanisms are lacking. 3. Does modulation of desensitization play a role in any components of neuromuscular blockade? 4. Can trapping of blocking agents in ion channels in some cases account for slowly reversible components of blockade? 5. Can closed-channel block provide an effective mechanism of neuromuscular block?
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The high affinity of the noncovalent interaction between biotin and streptavidin forms the basis for many diagnostic assays that require the formation of an irreversible and specific linkage between biological macromolecules. Comparison of the refined crystal structures of apo and a streptavidin:biotin complex shows that the high affinity results from several factors. These factors include the formation of multiple hydrogen bonds and van der Waals interactions between biotin and the protein, together with the ordering of surface polypeptide loops that bury the biotin in the protein interior. Structural alterations at the biotin binding site produce quaternary changes in the streptavidin tetramer. These changes apparently propagate through cooperative deformations in the twisted beta sheets that link tetramer subunits.
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