Search

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.

In this paper, we aim to measure Moroccan judicial efficiency and identify its determinants during the implementation of judicial reform. For this, we use a two-stage approach. First, we use data envelopment analysis (DEA) under output orientation to measure the efficiency of the 109 Moroccan courts. In the second stage, we explore determinants of efficiency using the Ordinary Least Squares regression model. The results show a low level of courts efficiency of 56.2% in 2018, despite an improvement during the reform implementation period. At the same time, the results show that efficiency increases in populated cities with high demands on justice, and in courts with high proportions of cases in process, and employing senior judges. Therefore, this paper presents some managerial implications for court managers and provides further research directions for assessing judicial efficiency.

The maintenance of a high density of the acetylcholine receptor (AChR) is the hallmark of the neuromuscular junction. Muscle-specific anchoring protein (αkap) encoded within the calcium/calmodulin-dependent protein kinase IIα (CAMK2A) gene is essential for the maintenance of AChR clusters both in vivo and in cultured muscle cells. The underlying mechanism by which αkap is maintained and regulated remains unknown. Here, using human cell lines, fluorescence microscopy, and pulldown and immunoblotting assays, we show that α-dystrobrevin (α-dbn), an intracellular component of the dystrophin glycoprotein complex, directly and robustly promotes the stability of αkap in a concentration-dependent manner. Mechanistically, we found that the phosphorylatable tyrosine residues of α-dbn are essential for the stability of α-dbn itself and its interaction with αkap, with substitution of three tyrosine residues in the α-dbn C terminus with phenylalanine compromising the αkap–α-dbn interaction and significantly reducing both αkap and α-dbn accumulation. Moreover, the αkap–α-dbn interaction was critical for αkap accumulation and stability. We also found that the absence of either αkap or α-dbn markedly reduces AChRα accumulation and that overexpression of α-dbn or αkap in cultured muscle cells promotes the formation of large agrin-induced AChR clusters. Collectively, these results indicate that the stability of αkap and α-dbn complex plays an important role in the maintenance of high-level expression of AChRs.

This paper focuses on measuring the efficiency and productivity change in 110 Moroccan courts between 2013 and 2018, which is the period of implementation of the judicial reform. The study also measures the technical and scale efficiency using the Data Envelopment Analysis method under output orientation. The Malmquist index was used to measure the productivity change decomposed into efficiency and technical change. The results show a low level of courts efficiency of 53.2%, with increasing returns to scale for most courts except administrative ones. The total factor productivity of courts shows an improvement of 6.3%, mainly due to technical change, except for the commercial courts whose productivity deteriorated during the study period. These results show the positive effect of the reform. Courts still have a margin for improving their efficiency by increasing inputs in order to benefit from economies of scale or by improving judges' productivity.

The Dystrophin Glycoprotein Complex (DGC) is a large multi-protein complex that links cytoskeleton actin to the extracellular matrix. This complex is critical in maintaining the structural integrity of muscle fibers and the stability of the neuromuscular synapse. The DGC consists of dystrophin and its utrophin homolog, as well as dystroglycans, sarcoglycans, sarcospan, syntrophins, and dystrobrevins. Deficiencies in DGC proteins result in several forms of muscular dystrophy with varying symptoms and degrees of severity in addition to structurally abnormal neuromuscular junctions (NMJs). This mini-review highlights current knowledge regarding the role of the DGC on the molecular dynamics of acetylcholine receptors (AChRs) as it relates to the formation and maintenance of the mammalian NMJ.

A novel mechanism is described for the agrin-mediated focal delivery of acetylcholine receptors (AChRs) to the postsynaptic membrane of the neuromuscular junction. Microtubule capture mediated by CLASP2 and its interaction partner, LL5β, and an intact subsynaptic actin cytoskeleton are both required for focal AChR transport to the synaptic membrane., 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 knockdown 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, knockdown of LL5β at the NMJ in vivo reduces the density and insertion of AChRs into the postsynaptic membrane. MT capture 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.

A 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. We show that rapsyn forms complex with both α-dystrobrevin and α-syntrophin at the crests of junctional folds. The linkage of rapsyn to α-syntrophin and/or α-dystrobrevin is mediated by utrophin, a protein localized at acetylcholine receptor (AChR)-rich domains. In mice deficient in α-syntrophin, in which utrophin is no longer present at the synapse, rapsyn interaction with α-dystrobrevin was completely abolished. This interaction was completely restored when either utrophin or α-syntrophin was introduced into muscles deficient in α-syntrophin. However, in neuromuscular junctions deficient in α-dystrobrevin, in which utrophin is retained, complex formation between rapsyn and α-syntrophin was unaffected. Using fluorescence recovery after photobleaching, we found that α-syntrophin turnover is 5-7 times faster than that of AChRs, and loss of α-dystrobrevin has no effect on rapsyn and α-syntrophin half-life, whereas the half-life of AChR was significantly altered. Altogether, these results provide new insights into the spatial distribution of dystrophin glycoprotein components and their dynamics in living mice.

Sarcopenia, the loss of muscle mass and strength during normal aging, involves coordinate changes in skeletal myofibers and the cells that contact them, including satellite cells and motor neurons. Here we show that the protein O-fucosyltransferase 1 gene (Pofut1), which encodes a glycosyltransferase required for NotchR-mediated cell-cell signaling, has reduced expression in aging skeletal muscle. Moreover, premature postnatal deletion of Pofut1 in skeletal myofibers can induce aging-related phenotypes in cis within skeletal myofibers and in trans within satellite cells and within motor neurons via the neuromuscular junction. Changed phenotypes include reduced skeletal muscle size and strength, decreased myofiber size, increased slow fiber (type 1) density, increased muscle degeneration and regeneration in aged muscles, decreased satellite cell self-renewal and regenerative potential, and increased neuromuscular fragmentation and occasional denervation. Pofut1 deletion in skeletal myofibers reduced NotchR signaling in young adult muscles, but this effect was lost with age. Increasing muscle NotchR signaling also reduced muscle size. Gene expression studies point to regulation of cell cycle genes, muscle myosins, NotchR and Wnt pathway genes, and connective tissue growth factor by Pofut1 in skeletal muscle, with additional effects on α dystroglycan glycosylation.

Intronic GGGGCC repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two major pathologies stemming from the hexanucleotide RNA expansions (HREs) have been identified in postmortem tissue: intracellular RNA foci and repeat-associated non-ATG dependent (RAN) dipeptides, although it is unclear how these and other hallmarks of disease contribute to the pathophysiology of neuronal injury. Here, we describe two novel lines of mice that overexpress either 10 pure or 102 interrupted GGGGCC repeats mediated by adeno-associated virus (AAV) and recapitulate the relevant human pathology and disease-related behavioural phenotypes. Similar levels of intracellular RNA foci developed in both lines of mice, but only mice expressing 102 repeats generated C9orf72 RAN pathology, neuromuscular junction (NMJ) abnormalities, dispersal of the hippocampal CA1, enhanced apoptosis, and deficits in gait and cognition. Neither line of mice, however, showed extensive TAR DNA-binding protein 43 (TDP-43) pathology or neurodegeneration. Our data suggest that RNA foci pathology is not a good predictor of C9orf72 RAN dipeptide formation, and that RAN dipeptides and NMJ dysfunction are drivers of C9orf72 disease pathogenesis. These AAV-mediated models of C9orf72-associated ALS/FTD will be useful tools for studying disease pathophysiology and developing new therapeutic approaches., Summary: C9orf72-linked motor neuron disease models with viral-mediated expression of GGGGCC repeat expansion in mice show neuropathology and behavioural deficits.

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.

WOS: 000415310800007 PubMed ID: 29144972 Science is ideally suited to connect people from different cultures and thereby foster mutual understanding. To promote international life science collaboration, we have launched "The Science Bridge'' initiative. Our current project focuses on partnership between Western and Middle Eastern neuroscience communities. Medical Research Council [MC_UP_1202/5]

Rapsyn, a 43 kDa scaffold protein, is required for the clustering of acetylcholine receptors (AChRs) at synaptic sites between mammalian motor neurons and muscle cells. However, the mechanism by which rapsyn is inserted and retained at postsynaptic sites at the neuromuscular junction (NMJ)in vivoremains largely unknown. We found that neither the N-terminal myristoylation nor the cysteine-rich RING H2 domain of rapsyn is required for its stable association with the postsynaptic membrane of NMJs. When N-myristoylation-defective rapsyn-EGFP mutant (G2A) and RING-H2 domain truncated rapsyn-EGFP were electroporated into sternomastoid muscles, a strong rapsyn fluorescent signal was observed selectively at synapses, similar to WT rapsyn-EGFP. The targeting of rapsyn-EGFP (WT and mutants) is independent of synaptic activity because they were inserted at denervated NMJs. However, when the coiled-coil domain (the AChR-binding domain of rapsyn) is deleted, rapsyn fails to associate with AChRs at NMJs of living mice. In cultured myoblasts (in which AChRs are absent), myristoylated WT rapsyn mostly localizes to lysosomes and is not associated with the plasma membrane. However, in the presence of AChR subunits, rapsyn molecules were targeted to the cell surface and formed aggregates with AChRs. The targeting of AChRs to the cell membrane, in contrast, does not require rapsyn because expressed AChRs are visible on the cell membranes of rapsyn-deficient myoblasts. These results provide evidence for an active role of AChRs in the targeting of rapsyn to the NMJin vivo.SIGNIFICANCE STATEMENTRapsyn is required for the clustering of acetylcholine receptors (AChRs) at postsynaptic sites. However, the mechanism by which rapsyn is targeted to synaptic sites at the vertebrate neuromuscular junction remains unclear. In this study, we showed that the coiled-coil domain of rapsyn is required for its targeting to the cell surface via its interaction with AChRs. In contrast, the targeting of AChRs to the cell membrane does not require rapsyn. These results indicate that AChRs play a critical role in the insertion and/or association of rapsyn with the plasma membrane of synaptic sites.

Neuregulin/ErbB signaling maintains high efficacy of synaptic transmission by stabilizing the postsynaptic apparatus via phosphorylation of α-dystrobrevin1., Neuregulin (NRG)/ErbB signaling is involved in numerous developmental processes in the nervous system, including synapse formation and function in the central nervous system. Although intensively investigated, its role at the neuromuscular synapse has remained elusive. Here, we demonstrate that loss of neuromuscular NRG/ErbB signaling destabilized anchoring of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane and that this effect was caused by dephosphorylation of α-dystrobrevin1, a component of the postsynaptic scaffold. Specifically, in mice in which NRG signaling to muscle was genetically or pharmacologically abolished, postsynaptic AChRs moved rapidly from the synaptic to the perisynaptic membrane, and the subsynaptic scaffold that anchors the AChRs was impaired. These defects combined compromised synaptic transmission. We further show that blockade of NRG/ErbB signaling abolished tyrosine phosphorylation of α-dystrobrevin1, which reduced the stability of receptors in agrin-induced AChR clusters in cultured myotubes. Our data indicate that NRG/ErbB signaling maintains high efficacy of synaptic transmission by stabilizing the postsynaptic apparatus via phosphorylation of α-dystrobrevin1.

α-Syntrophin (α-syn), a scaffold protein, links signaling molecules to the dystrophin–glycoprotein complex. Absence of α-syn from the DGC is known to lead to structurally aberrant neuromuscular junctions (NMJs) with few acetylcholine receptors (AChRs) clustered at synaptic sites. Using α-syn knock-out mice, we show that during the first postnatal week, α-syn is not required for synapse formation. However, at postnatal day 6 (P6)–P7, the structural integrity of the postsynaptic apparatus is altered, the turnover rate of AChRs increases significantly, and the number/density of AChRs is impaired. At the adult α-syn−/−NMJ, the turnover rate of AChRs is ∼4 times faster than wild-type synapses, and most removed receptors are targeted to degradation as few AChRs recycled to synaptic sites. Biochemical analyses show that in muscle cells of adult knock-out α-syn mice, total AChRs and scaffold protein rapsyn are significantly reduced, the 89 kDa and 75 kDa isoforms of tyrosine phosphorylated α-dystrobrevin (α-dbn) 1 (which are required for the maintenance and stability of AChR in α-dbn−/−synapses) are barely detectable. Electroporation of GFP-α-dbn1 in α-syn−/−muscle cells partially restored receptor density, turnover rate, and the structural integrity of the postsynaptic apparatus, whereas expression of rapsyn-GFP failed to rescue the α-syn−/−synaptic phenotype. These results demonstrate that α-syn is required for the maturation and stability of the postsynaptic apparatus and suggest that α-syn may act via α-dbn1.

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 synapsesin 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.

At the peripheral neuromuscular junction (NMJ), a significant number of nicotinic acetylcholine receptors (AChRs) recycle back into the postsynaptic membrane after internalization to intermingle with not-yet-internalized`pre-existing' AChRs. However, the way in which these receptor pools are maintained and regulated at the NMJ in living animals remains unknown. Here,we demonstrate that recycled receptors in functional synapses are removed approximately four times faster than pre-existing receptors, and that most removed recycled receptors are replaced by new recycled ones. In denervated NMJs, the recycling of AChRs is significantly depressed and their removal rate increased, whereas direct muscle stimulation prevents their loss. Furthermore,we show that protein tyrosine phosphatase inhibitors cause the selective accumulation of recycled AChRs in the peri-synaptic membrane without affecting the pre-existing AChR pool. The inhibition of serine/threonine phosphatases,however, has no effect on AChR recycling. These data show that recycled receptors are remarkably dynamic, and suggest a potential role for tyrosine dephosphorylation in the insertion and maintenance of recycled AChRs at the postsynaptic membrane. These findings may provide insights into long-term recycling processes at less accessible synapses in the central nervous system in vivo.

For synapses to form and function, neurotransmitter receptors must be recruited to a location on the postsynaptic cell in direct apposition to presynaptic neurotransmitter release. However, once receptors are inserted into the postsynaptic membrane, they are not fixed in place but are continually exchanged between synaptic and extrasynaptic regions, and they cycle between the surface and intracellular compartments. This article highlights and compares the current knowledge about the dynamics of acetylcholine receptors at the vertebrate peripheral neuromuscular junction and AMPA, N-methyl-D-aspartate, and gamma-aminobutyric acid receptors in central synapses.

The efficiency of synaptic transmission between nerve and muscle depends on the number and density of acetylcholinesterase molecules (AChE) at the neuromuscular junction. However, little is known about the way this density is maintained and regulated in vivo. By using time lapse and quantitative fluorescence imaging assays in living mice, we demonstrated that insertion of new AChEs occurs within hours of saturating pre-existing AChEs with fasciculin2, a snake toxin that selectively labels AChE. In the absence of muscle postsynaptic activity or evoked nerve presynaptic neurotransmitter release, AChE insertion was decreased significantly, whereas direct stimulation of the muscle completely restored AChE insertion to control levels. This activity-dependent AChE insertion is mediated by intracellular calcium. In muscle stimulated in the presence of a Ca2+ channel blocker or calcium-permeable Ca2+ chelator, AChE insertion into synapses was significantly decreased, whereas ryanodine or ionophore A12387 treatment of blocked and unstimulated synapses significantly increased AChE insertion. These results demonstrated that synaptic activity is critical for AChE insertion and indicated that a rise in intracellular calcium either through voltage-gated calcium channels or from intracellular stores is critical for proper AChE insertion into the adult synapse.

α-Dystrobrevin (DB), a cytoplasmic component of the dystrophin–glycoprotein complex, is found throughout the sarcolemma of muscle cells. Mice lacking αDB exhibit muscular dystrophy, defects in maturation of neuromuscular junctions (NMJs) and, as shown here, abnormal myotendinous junctions (MTJs). In normal muscle, alternative splicing produces two main αDB isoforms, αDB1 and αDB2, with common NH2-terminal but distinct COOH-terminal domains. αDB1, whose COOH-terminal extension can be tyrosine phosphorylated, is concentrated at the NMJs and MTJs. αDB2, which is not tyrosine phosphorylated, is the predominant isoform in extrajunctional regions, and is also present at NMJs and MTJs. Transgenic expression of either isoform in αDB−/− mice prevented muscle fiber degeneration; however, only αDB1 completely corrected defects at the NMJs (abnormal acetylcholine receptor patterning, rapid turnover, and low density) and MTJs (shortened junctional folds). Site-directed mutagenesis revealed that the effectiveness of αDB1 in stabilizing the NMJ depends in part on its ability to serve as a tyrosine kinase substrate. Thus, αDB1 phosphorylation may be a key regulatory point for synaptic remodeling. More generally, αDB may play multiple roles in muscle by means of differential distribution of isoforms with distinct signaling or structural properties.

Rapsyn, a scaffold protein, is required for the clustering of acetylcholine receptors (AChRs) at contacts between motor neurons and differentiating muscle cells. Rapsyn is also expressed in cells that do not express AChRs. However, its function in these cells remains unknown. Here, we showed that rapsyn plays an AChR-independent role in organizing the distribution and mobility of lysosomes. In cells devoid of AChRs, rapsyn selectively induced the clustering of lysosomes at high density in the juxtanuclear region without affecting the distribution of other intracellular organelles. However, when the same cells overexpress AChRs, rapsyn is recruited away from lysosomes to co-localize with AChR clusters on the cell surface. In rapsyn-deficient (rapsyn−/−) myoblasts or cells overexpressing rapsyn mutants, lysosomes are scattered within the cell and highly dynamic. The increased mobility of lysosomes in rapsyn−/− cells is associated with a significant increase of lysosomal exocytosis as evidenced by increased release of lysosomal enzymes and the plasma membrane damage when cells were challenged with the bacterial pore-forming toxin streptolysin-O. These findings uncover a new link between rapsyn, lysosome positioning, exocytosis, and plasma membrane integrity.

We show that fluorescently tagged ligands with high affinity for their targets can be reversibly unbound by focused laser excitation. By sequential unbinding and relabeling with different colors of alpha-bungarotoxin, we selectively labeled adjacent pools of acetylcholine receptors (AChRs) at neuromuscular junctions of adult mice. Timelapse imaging in vivo revealed that synaptic AChRs completely intermingle over approximately 4 days and many extrasynaptic AChRs are incorporated into the synapse each day. In mice that lacked alpha-dystrobrevin, a component of the dystrophin-glycoprotein complex, rates of AChR turnover, and intermingling were increased approximately 4- to 5-fold. These results demonstrate remarkable molecular dynamism underlying macroscopic stability of the postsynaptic membrane, and establish alpha-dystrobrevin as a key control point for regulation of mobility and turnover.

Amyloid precursor protein (APP), associated with Alzheimer’s disease plaques, is known to be present in synapses of the brain and in the adult neuromuscular junction (NMJ). In the present study we examined protein and gene expression of APP during the development of mouse skeletal muscle. Using immunocytochemical approaches, we found that APP is first detected in myotube cytoplasm at embryonic day 16 and becomes progressively concentrated at the NMJ beginning at birth until adulthood. The colocalization between APP and acetylcholine receptors at the NMJ is only partial at birth, but becomes complete upon reaching adulthood. We observed that all APP isoforms, including the Kunitz-containing (protease inhibitor or KPI) forms, are up-regulated from birth to postnatal day 5 and then decreased to reach the low levels observed in the adult. This suggests the involvement of APP during the events which lead to a mature mono-innervated synapse. A 92-kDa band, characteristic of a cleaved APP695 isoform and not due to a new muscle-specific alternative spliced form, was observed from postnatal day 15 following completion of polyneuronal synapse elimination. Taken together, these data suggest that skeletal muscle APP may well play a role in the differentiation of skeletal muscle and in the formation and maturation of NMJs.

Quantitative fluorescence imaging was used to study the regulation of acetylcholine receptor (AChR) number and density at neuromuscular junctions in living adult mice. At fully functional synapses, AChRs have a half-life of about 14 days. However, 2 hours after neurotransmission was blocked, the half-life of the AChRs was now less than a day; the rate was 25 times faster than before. Most of the lost receptors were not quickly replaced. Direct muscle stimulation or restoration of synaptic transmission inhibited this process. AChRs that were removed from nonfunctional synapses resided for hours in the perijunctional membrane before being locally internalized. Dispersed AChRs could also reaggregate at the junction once neurotransmission was restored. The rapid and reversible alterations in AChR density at the neuromuscular junction in vivo parallel changes thought to occur in the central nervous system at synapses undergoing potentiation and depression.

During vertebrate neuromuscular development, all muscle fibers are transiently innervated by more than one neuron. Among the numerous factors shown to potentially influence the passage from poly- to mononeuronal innervation, serine proteases and their inhibitors appear to play important roles. In this regard, protease nexin I (PNI), a potent inhibitor of the serine protease, thrombin, is highly localized to the neuromuscular junction (NMJ). In turn, thrombin is responsible for activity-dependent synapse elimination both in an in vitro model, and in vivo. In the present study, we used a monospecific anti-PNI polyclonal antibody to study the developmental kinetics of PNI expression in mouse leg skeletal muscle. By using immunoblotting, we detected PNI at embryonic day 16 (E16), as a 48-kDa band. This 48-kDa PNI band became prominent in leg muscle extracts at postnatal day 5 (P5) and remained so in extracts from adult muscle. In contrast, a higher molecular weight immunoreactive PNI band, which was sodium dodecyl sulfate– and β-mercaptoethanol–resistant, was first detected at E16, increased at birth (P0), and then decreased at P15, i.e., after the wave of polyneuronal synapse elimination had occurred in these muscles. The results of an enzyme-linked immunosorbent assay, measuring active, complexed, and truncated PNI, correlated with Western blot data. We used immunocytochemistry to probe the localization of PNI at the NMJ and found that PNI was present in the cytoplasm of myotubes at E16, but neither then nor at birth did it colocalize with acetylcholine receptors. PNI became localized at NMJs by P5 and increased by P15, after which it remained stably concentrated there in the adult. Finally, we studied the gene expression of PNI mRNA, by using Northern blotting, and showed that PNI mRNA was present in skeletal muscle and remained stable throughout the time-course studies, suggesting that developmental regulation of muscle PNI occurs principally at the translational and/or post-translational levels. These results suggest that the localization of PNI, through a binding site or “receptor” may play an important role in differentiation and maintenance of synapse. J. Comp. Neurol. 397:572–579, 1998. © 1998 Wiley-Liss, Inc.

The mature neuromuscular junction (NMJ) is the best characterized cholinergic synapse. The maintenance of a high number and density of nicotinic acetylcholine receptors (nAChRs) at the postsynaptic membrane adjacent to the nerve terminal are crucial for NMJ function. This density is maintained by several factors, ranging from synaptic activity to postsynaptic scaffold proteins. Decreases in postsynaptic nAChR density are related to myasthenic syndromes in the peripheral NMJ, but are also associated in central synapses with neurodegenerative diseases such as Alzheimer’s. In this review, we focus particularly on our increasing knowledge about the molecular dynamics of nAChR at the peripheral cholinergic NMJ and their regulation by the postsynaptic proteins of the dystrophin glycoprotein complex (DGC).

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.

The balance of serine proteases and inhibitors in nerve and muscle is altered during programmed- and injury-induced remodeling. A serpin, alpha 1-antichymotrypsin (alpha 1-ACT), and Kunitz-inhibitor containing forms of the beta-amyloid precursor protein (beta APP) may be important components of this balance. In the present study, we analyzed their expression in primary cultures of human myogenic (satellite) cells that mimic myogenic differentiation using Western blotting and immunocytochemistry. In vitro results were compared to in vivo results from normal adult human skeletal muscle biopsies. Using an anti-alpha 1-ACT polyclonal antibody, we detected a 62 kDa immunoreactive band both in cultured human myogenic cells (mononucleated myoblasts as well as multi-nucleated myotubes) and in extracts of human muscle biopsies. With a polyclonal anti-beta APP antibody we found two bands (105 and 120 kDa) in myoblasts and myotubes in culture. However, the same antibody recognized only a single band at 92 kDa in biopsies. By immunocytochemistry, both alpha 1-ACT and beta APP were indistinctly present on localized to the surface of myoblasts in culture. In contrast, these inhibitors were dense on myotube surfaces, where they often formed distinct aggregates and frequently co-localized. In permeabilized muscle cells, alpha 1-ACT and beta APP appeared to be localized to the perikarya of both myoblasts and myotubes. Confirming previous results, both alpha 1-ACT and beta APP were present at the neuromuscular junction in human muscle sections. These developmental changes found during in vitro myogenesis for alpha 1-ACT and beta APP, both serine protease inhibitors, reinforce the hypothesis that regulation of the serine proteases and serine protease inhibitors plays an important role in neuromuscular differentiation.

Recombinant human insulin-like growth factor-I (IGF-I) is being considered as a possible therapeutic agent for the treatment of motoneuron diseases like amyotrophic lateral sclerosis. The neurological mutant mouse wobbler, carries an autosomal recessive gene (wr) and has been characterized as a model of lower motoneuron disorders with associated muscle atrophy, denervation and reinnervation. The purpose of the present study was to determine the possible beneficial effect of IGF-I administration in this mouse model. Upon diagnosis at 4 weeks of age, affected mice and their control normal littermates received human recombinant IGF-I (1 mg/kg) or vehicle solution, once a day, for 6 weeks. Body weight and grip strength were evaluated periodically during the treatment period. Mean muscle fiber diameter on biceps brachii sections, choline acetyltransferase activity in muscle extracts, and motoneuron numbers in spinal cord sections were determined. IGF-I treated wobbler mice showed a marked weight increase from 3 to 6 weeks of treatment in comparison with placebo treated mutant mice. At the end of the treatment, grip strength, estimated by dynamometer resistance, was 40% higher in IGF-I treated versus placebo treated animals. Mean muscle fiber diameter which is smaller in wobbler mice than in normal mice was increased in IGF-I treated mutants. However, in this study the muscle choline acetyltransferase activity and the number of spinal cord motoneurons were unchanged. Thus, IGF-I administration mainly results in a significant effect on the behavioral and skeletal muscle histochemical parameters of the wobbler mouse mutant.

Kunitz-inhibitor containing forms of the beta-amyloid precursor protein (beta APP), known also as protease nexin II (PNII), and alpha 1-antichymotrypsin (alpha 1-ACT), a serpin, are important components of the serine protease and inhibitor balance in many tissues. In the nervous system, this balance may have trophic or growth factor activity at different stages of development, after injury and in disease states. In the current study, using immunocytochemistry and Western blotting with antibodies against the human homologues, we analyzed whether denervation affected the localization of beta APP and alpha 1-ACT in adult mouse muscle following axotomy. In mouse muscle, anti-human alpha 1-ACT antibody detected a 60 kD immunoreactive band and anti-human beta APP antibody a band at 92 kD in both normal and denervated extracts. beta APP was present in normal mouse muscle at both neuromuscular junctions and within intramuscular nerves. alpha 1-ACT was also detected at neuromuscular junctions, on the perineurium and endothelial cell surfaces. Following axotomy, both beta APP and alpha 1-ACT disappeared from intramuscular nerves simultaneously. However, at the neuromuscular junction, alpha 1-ACT decreased more rapidly with beta APP lingering before disappearing. Since both alpha 1-ACT as well as beta APP are present within senile plaques in Alzheimer's disease brains such experiments with the nicotinic, cholinergic neuromuscular synapse in denervated muscle may help to focus experiments on the mechanism of synapse loss as well as plaque deposition in this disease.

Our article documents recent studies in the proteolytic processing of the Alzheimer's beta-amyloid protein precursor (beta-APP), as well as the role of thrombin and its potent inhibitor, protease nexin I in Alzheimer's disease (AD). Since synapse loss correlates best with cognitive decline in AD, we also present in detail, our model of synapse formation and elimination, reviewing recent findings related to the subject as well as our own original data. Recent exciting findings concerning the involvement of thrombin-like activity in synapse elimination, which we feel to be important in neural plasticity are also discussed.

At the mammalian skeletal neuromuscular junction, cycling of nicotinic ACh receptors (nAChRs) is critical for the maintenance of a high postsynaptic receptor density. However, the mechanisms that regulate nAChRs recycling in living animals remain unknown. Usingin vivotime-lapse imaging, fluorescence recovery after photobleaching, and biochemical pull down assays, we demonstrated that recycling of internalized nAChRs into fully functional and denervated synapses was promoted by both direct muscle stimulation and pharmacologically induced intracellular calcium elevations. Most of internalized nAChRs are recycled directly into synaptic sites. Chelating of intracellular calcium below resting level drastically decreased cycling of nAChRs. Furthermore we found that calcium-dependent AChR recycling is mediated by Ca2+/calmodulin-dependent kinase II (CaMKII). Inhibition of CaMKII selectively blocked recycling and caused intracellular accumulation of internalized nAChRs, whereas internalization of surface receptors remained unaffected. Electroporation of CaMKII-GFP isoforms into the sternomastoid muscle showed that muscle-specific CaMKIIβm isoform is highly expressed at the neuromuscular junction (NMJ) and precisely colocalized with nAChRs at crests of synaptic folds while the CaMKIIγ and δ isoforms are poorly expressed in synaptic sites. These results indicate that Ca2+along with CaMKII activity are critical for receptor recycling and may provide a mechanism by which the postsynaptic AChR density is maintained at the NMJin vivo.

The efficacy of synaptic transmission depends on the maintenance of a high density of neurotransmitter receptors and their associated scaffold proteins in the postsynaptic membrane. While the dynamics of receptors has been extensively studied, the dynamics of the intracellular scaffold proteins that make up the postsynaptic density are largely unknownin vivo. Here, we focused on the dynamics of rapsyn, a protein required for the clustering and maintenance of acetylcholine receptor (AChR) density at postsynaptic sites. Using time-lapse imaging, we demonstrated that rapsyn is remarkably dynamic compared to AChRs at functional synapses, turning over 4–6 times more rapidly than AChRs. In addition we found that the rapid turnover of rapsyn is insensitive to alterations in synaptic activity, whereas AChR turnover is profoundly affected, illustrating that rapsyn and receptor dynamics are controlled by distinct mechanisms. These data indicate that individual postsynaptic components are in permanent exchange despite the overall stability of synaptic structure, which may play a role in synaptic plasticity.

Changes in synaptic strength are important for synaptic development and synaptic plasticity. Most directly responsible for these synaptic changes are alterations in synaptic receptor number and density. Although alterations in receptor density mediated by the insertion, lateral mobility, removal, and recycling of receptors have been extensively studied, the dynamics and regulators of intracellular scaffolding proteins have only recently begun to be illuminated. In particular, a closer look at the receptor-associated proteins, which bind to receptors and are necessary for their synaptic localization and clustering, has revealed broader functions than previously thought and some rather unexpected thematic similarities. More than just “placeholders” or members of a passive protein “scaffold,” receptor-associated proteins in every synapse studied have been shown to provide a number of signaling roles. In addition, the most recent state-of-theart imaging has revealed that receptor-associated proteins are highly dynamic and are involved in regulating synaptic receptor density. Together, these results challenge the view that receptor-associated proteins are members of a static and stable scaffold and argue that their dynamic mobility may be essential for regulating activity-dependent changes in synaptic strength.—Bruneau, E. G., Esteban, J. A., Akaaboune, M. Receptorassociated proteins and synaptic plasticity. FASEB J. 23, 679–688 (2009), This work was supported by the U.S. National Institutes of Health (M.A.) and a National Research Service Award (G.B.), the National Science Foundation (M.A.), and the Muscular Dystrophy Association (M.A.).

Summary The maintenance of a high density of postsynaptic receptors is essential for proper synaptic function. At the neuromuscular junction, acetylcholine receptor (AChR) aggregation is induced by nerve-clustering factors and mediated by scaffolding proteins [1]. Although the mechanisms underlying AChR clustering have been extensively studied [2–5], the role that the receptors themselves play in the clustering process and how they are organized with scaffolding proteins is not well understood. Here, we report that the exposure of AChRs labeled with Alexa 594 conjugates to relatively low-powered laser light caused an effect similar to chromaphore-assisted light inactivation (CALI) [6, 7], which resulted in the unexpected dissipation of the illuminated AChRs from clusters on cultured myotubes. This technique enabled us to demonstrate that AChR removal from illuminated regions induced the removal of scaffolding proteins and prevented the accumulation of new AChRs and associated scaffolding proteins. Further, the dissipation of clustered AChRs and scaffold was spatially restricted to the illuminated region and had no effect on neighboring nonilluminated AChRs. These results provide direct evidence that AChRs are essential for the local maintenance and accumulation of intracellular scaffolding proteins and suggest that the scaffold is organized into distinct modular units at AChR clusters.

Acetylcholinesterase (AChE) is an enzyme that terminates acetylcholine neurotransmitter function at the synaptic cleft of cholinergic synapses. However, the mechanism by which AChE number and density are maintained at the synaptic cleft is poorly understood. In this work, we used fluorescence recovery after photobleaching, photo-unbinding, and quantitative fluorescence imaging to investigate the surface mobility and stability of AChE at the adult innervated neuromuscular junction of living mice. In wild-type synapses, we found that nonsynaptic (perisynaptic and extrasynaptic) AChEs are mobile and gradually recruited into synaptic sites and that most of the trapped AChEs come from the perijunctional pool. Selective labeling of a subset of synaptic AChEs within the synapse by using sequential unbinding and relabeling with different colors of streptavidin followed by time-lapse imaging showed that synaptic AChEs are nearly immobile. At neuromuscular junctions of mice deficient in α-dystrobrevin, a component of the dystrophin glycoprotein complex, we found that the density and distribution of synaptic AChEs are profoundly altered and that the loss rate of AChE significantly increased. These results demonstrate that nonsynaptic AChEs are mobile, whereas synaptic AChEs are more stable, and that α-dystrobrevin is important for controlling the density and stability of AChEs at neuromuscular synapses.

Rapsyn, a cytoplasmic receptor-associated protein, is required for the clustering of acetylcholine receptors (AChRs). Although AChR dynamics have been extensively studied, little is known about the dynamics of rapsyn. Here, we used a rapsyn-green fluorescent protein (GFP) fusion protein and quantitative fluorescent imaging to study the dynamics of rapsyn in transfected C2C12 myotubes. First, we found that rapsyn-GFP expression at clusters did not alter AChR aggregation, function, or turnover. Quantification of rapsyn immunofluorescence indicated that the expression of rapsyn-GFP proteins at clusters does not increase the overall rapsyn density compared with untransfected myotube clusters. Using time lapse imaging and fluorescence recovery after photobleaching, we demonstrated that the recovery of rapsyn-GFP fluorescence at clusters was very fast, with a halftime of about approximately 1.5 h (approximately 3 times faster than AChRs). Inhibition of protein kinase C significantly altered receptor insertion, but it had no effect on rapsyn insertion. When cells were treated with the broad spectrum kinase inhibitor staurosporine, receptor insertion was decreased even further. However, inhibition of protein kinase A had no effect on insertion of either rapsyn or receptors. Finally, when cells were treated with neural agrin, rapsyn and AChRs were both directed away from preexisting clusters and accumulated together in new small clusters. These results demonstrate the remarkable dynamism of rapsyn, which may underlie the stability and maintenance of the postsynaptic scaffold and suggest that the insertion of different postsynaptic proteins may be operating independently.

At cholinergic synapses, acetylcholinesterase (AChE) is critical for ensuring normal synaptic transmission. However, little is known about how this enzyme is maintained and regulated in vivo. In this work, we demonstrate that the dissociation of fluorescently-tagged fasciculin 2 (a specific and selective peptide inhibitor of AChE) from AChE is extremely slow. This fluorescent probe was used to study the removal and insertion of AChE at individual synapses of living adult mice. After a one-time blockade of AChEs with fluorescent fasciculin 2, AChEs are removed from synapses initially at a faster rate (t(1/2) of approximately 3 days) and later at a slower rate (t(1/2) of approximately 12 days). Most of the removed AChEs are replaced by newly inserted AChEs over time. However, when AChEs are continuously blocked with fasciculin 2, the removal rate increases substantially (t(1/2) of approximately 12 h), and most of the lost AChEs are not replaced by newly inserted AChE. Furthermore, complete one-time inactivation of AChE activity significantly increases the removal of postsynaptic nicotinic acetylcholine receptors (AChRs). Finally, time lapse imaging reveals that synaptic AChEs and AChRs that are removed from synapses are co-localized in the same pool after being internalized. These results demonstrate a remarkable AChE dynamism and argue for a potential link between AChE function and postsynaptic receptor lifetime.

Using optical imaging assays, we investigated the dynamics of acetylcholine receptors (AChRs) at laminin-associated clusters on cultured myotubes in the absence or presence of the nerve-derived clustering factor, agrin. Using fluorescence recovery after photobleaching (FRAP) on fluorescent bungarotoxin-labeled receptors, we found that approximately 9% of original fluorescence was recovered after 8 h as surface AChRs were recruited into clusters. By quantifying the loss of labeled receptors and the recovery of fluorescence after photobleaching, we estimated that the half-life of clustered receptors was approximately 4.5 h. Despite the rapid removal of receptors, the accumulation of new receptors at clusters was robust enough to maintain receptor density over time. We also found that the AChR half-life was not affected by agrin despite its role in inducing the aggregation of AChRs. Interestingly, when agrin was added to myotubes grown on laminin-coated substrates, most new receptors were not directed into preexisting laminin-induced clusters but instead formed numerous small aggregates on the entire muscle surface. Time-lapse imaging revealed that the agrin-induced clusters could be seen as early as 1 h, and agrin treatment resulted in the complete dissipation of laminin-associated clusters by 24 h. These results reveal that while laminin and agrin are involved in the clustering of receptors they are not critical to the regulation of receptor metabolic stability at these clusters, and further argue that agrin is able to rapidly and fully negate the laminin substrate clustering effect while inducing the rapid formation of new clusters.

alpha-Dystrobrevin (DB), a cytoplasmic component of the dystrophin-glycoprotein complex, is found throughout the sarcolemma of muscle cells. Mice lacking alphaDB exhibit muscular dystrophy, defects in maturation of neuromuscular junctions (NMJs) and, as shown here, abnormal myotendinous junctions (MTJs). In normal muscle, alternative splicing produces two main alphaDB isoforms, alphaDB1 and alphaDB2, with common NH2-terminal but distinct COOH-terminal domains. alphaDB1, whose COOH-terminal extension can be tyrosine phosphorylated, is concentrated at the NMJs and MTJs. alphaDB2, which is not tyrosine phosphorylated, is the predominant isoform in extrajunctional regions, and is also present at NMJs and MTJs. Transgenic expression of either isoform in alphaDB-/- mice prevented muscle fiber degeneration; however, only alphaDB1 completely corrected defects at the NMJs (abnormal acetylcholine receptor patterning, rapid turnover, and low density) and MTJs (shortened junctional folds). Site-directed mutagenesis revealed that the effectiveness of alphaDB1 in stabilizing the NMJ depends in part on its ability to serve as a tyrosine kinase substrate. Thus, alphaDB1 phosphorylation may be a key regulatory point for synaptic remodeling. More generally, alphaDB may play multiple roles in muscle by means of differential distribution of isoforms with distinct signaling or structural properties.

We addressed the balance between thrombin and its serpin protease nexin I (PNI) after sciatic nerve injury in the mouse. Prothrombin levels increased twofold 24 h after nerve crush, as measured by a specific chromogenic assay, and peaked at day 3. Thrombin activity also increased 2–4 days after injury in distal sciatic nerve segments. Nerve RNA analysis using reverse transcriptase-polymerase chain reaction (RT-PCR) assay confirmed that prothrombin was synthesized locally. We also monitored PNI levels in these injured nerve samples by complex formation with an 125I-labeled target protease and found peak activity occurring later, 6–9 days after the thrombin induction. These data indicate that nerve injury first induces the synthesis of prothrombin, which is subsequently converted to active thrombin. Nerve crush-induced thrombin is followed by the generation of functionally active PNI and may be directly responsible for its induction. By immunocytochemistry with anti-PNI antibody, we found that activated Schwann cells were the source of induced PNI. These results support the concept that the balance between serine proteases and their serpins is dysregulated during nerve injury and suggests a role for its reestablishment in nerve damage repair.

Protease nexin I (PNI) is a 43- to 50-kDa glycoprotein capable of inhibiting a number of serine proteases and belongs to the serpin superfamily. PNI is identical to glia-derived nexin, a neurite outgrowth promoter by virtue of its thrombin-inhibiting activity. Of particular relevance to neuromuscular biology and pathology, PNI was the first serpin shown to be highly localized to the neuromuscular junction and it maps to precisely the same locus as autosomal recessive amyotrophic lateral sclerosis (ALSJ) at chromosome 2q33–35. In the present report, we now show that in cultures of human skeletal muscle, PNI protein is expressed only after myoblast fusion into multinuclear myotubes and is localized in patches on their surfaces. We performed complex formation experiments with labeled thrombin, another target protease for PNI, with intact human muscle cells in culture. We detected specific SDS-stable PNI/thrombin complexes in myotube extracts only, indicating that active PNI was bound to their surfaces. We studied the gene expression of PNI mRNA using a 300-bp cDNA synthesized from the published sequence of human PNI. Confirming the protein data, upregulation of PNI appears in myotubes using Northern blot analysis. The current results reinforce the hypothesis that the regulation of the balance of serine proteases and serpins, such as PNI, is involved in muscle differentiation. They also prompt us to explore PNI abnormalities in several neuromuscular diseases, including ALSJ.

A serpin, alpha 1-antichymotrypsin (alpha 1-ACT), and Kunitz inhibitor containing forms of the beta-amyloid precursor protein (beta APP) may be important components of the balance between serine proteases and inhibitors in the nervous system. In the current report we studied whether axotomy affected the localization of beta APP and alpha 1-ACT in adult mouse muscle. Immunocytochemical experiments indicated that beta APP was present in normal muscle both at neuromuscular junctions and within intramuscular nerves. alpha 1-ACT was also present at neuromuscular junctions, on the perineurium of nerves and endothelial cell surfaces. Following axotomy, both beta APP and alpha 1-ACT disappeared from intramuscular nerves simultaneously. However, at the neuromuscular junction alpha 1-ACT decreased more rapidly with beta APP lingering before disappearing.

Apolipoprotein E (ApoE)-epsilon 4 allele has been associated with late onset familial Alzheimer's disease (AD). In both familial and sporadic AD brain, ApoE is localized to the vessel walls, senile amyloid plaques, and neurofibrillary tangles. ApoE is also an 'injury-response' macromolecule in peripheral nerves and was reported to increase in response to injury. We have demonstrated that Alzheimer beta-amyloid precursor protein and a serpin alpha 1-antichymotrypsin also found accumulated in senile plaques in AD brain, were also localized at neuromuscular junctions (NMJs). Using immunocytochemistry, our present results indicate that ApoE is found in normal mouse, rat and human skeletal muscle and concentrated at the NMJs as it is in the senile plaques in AD brain. Such experiments may shed light on the mechanism of synapse loss, as well as plaque formation in this neurodegenerative disease.