Number Of Acetylcholine Receptor Clusters Research Articles

Decreased mRNA Expression of PGC-1α and PGC-1α-Regulated Factors in the SOD1G93AALS Mouse Model and in Human Sporadic ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motoneuron loss. Although the cause of ALS is unknown, oxidative stress, inflammation, and mitochondrial dysfunction have been identified as important components of its pathogenesis. Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) plays a central role in the regulation of mitochondrial metabolism and biogenesis via activation of transcription factors, such as nuclear respiratory factors 1 and 2 and mitochondrial transcription factor A (Tfam). Alterations in PGC-1α expression and function have previously been described in models of Huntington and Alzheimer diseases. Moreover, the protective effects of PGC-1α have been shown in animal models of ALS. Levels of PGC-1α correlate with the number of acetylcholine receptor clusters in muscle. This is of particular interest because neurodegeneration in ALS may be a dying-back process. We investigated mRNA and protein expressions of PGC-1α and PGC-1α-regulated factors in the spinal cord and muscle tissues of SOD1 ALS mice and in ALS patients. We detected significant alterations in mRNA expression of PGC-1α and downstream factors with their earliest occurrence in muscle tissue. Our data provide evidence for a role of PGC-1α in mitochondrial dysfunction both in the ALS mouse model and in human sporadic ALS that is probably most relevant in the skeletal muscle.

Nestin Is Not Essential for Development of the CNS But Required for Dispersion of Acetylcholine Receptor Clusters at the Area of Neuromuscular Junctions

Nestin is expressed in many different progenitors during development including those of the CNS, heart, skeletal muscle, and kidney. The adult expression is mainly restricted to the subependymal zone and dentate gyrus of the brain, the neuromuscular junction, and renal podocytes. In addition, this intermediate filament protein has served as a marker of neural stem/progenitor cells for close to 20 years. Therefore it is surprising that its function in development and adult physiology is still poorly understood. Here we report that nestin deficiency is compatible with normal development of the CNS. The mutant mice, however, show impaired motor coordination. Furthermore, we found that the number of acetylcholine receptor clusters, the nerve length, and the endplate bandwidth are significantly increased in neuromuscular junction area of nestin-deficient mice. This is similar to the phenotype described for deficiency of cyclin-dependent kinase 5 (Cdk5), a candidate downstream affecter of nestin. Moreover, we demonstrate that nestin deficiency can rescue maintenance of acetylcholine receptor clusters in the absence of agrin, similar to Cdk5/agrin double knock-outs, suggesting that the observed nestin deficiency phenotype is the consequence of aberrant Cdk5 activity.

Structure of neuromuscular junctions and differentiation of striated muscle fibers in mdx mice after bone-marrow stem cell therapy

Mdx mice are a model of Duchenne muscular dystrophy caused by deficiency of dystrophin. Muscles of mdx mice are characterized by high levels of striated muscle fibers death and, accordingly, by a high level of its regeneration. Moreover, the structure of neuromuscular junctions in mdx mice is altered. Changes in the structure of mdx mice neuromuscular junctions against a background of increasing differentiation of striated muscle fibers after C57BL/6 Lin (-) bone marrow stem cells transplantation were investigated. The muscles were studied in 4, 8, 16 and 24 weeks after transplantation. We observed that the level of striated muscle fibers loss was decreased from the 4th week after transplantation of bone marrow stem cells. Accumulation of muscle fibers without centrally located nuclei began from the 8th week, and dystrophin synthesis was increased at the 16th and 24th weeks after bone marrow stem cells transplantation. Longitudinal sections of quadriceps muscles of mdx mice showed decrease in the number of acetylcholine receptors clusters in neuromuscular junctions and a simultaneous increase in acetylcholine receptor clusters area during the 4th week after transplantation. In 16 weeks after bone marrow stem cells transplantation, total neuromuscular junction area was increased due to increase in the area of acetylcholine receptors clusters and to increase in their number as well. Thus, single intramuscular transplantation of C57BL/6 Lin (-) bone marrow stem cells induces an increase in differentiation of mdx mice striated muscle fibers and improves the structure of neuromuscular junctions.

Wnt/β-Catenin Signaling Suppresses Rapsyn Expression and Inhibits Acetylcholine Receptor Clustering at the Neuromuscular Junction

The dynamic interaction between positive and negative signals is necessary for remodeling of postsynaptic structures at the neuromuscular junction. Here we report that Wnt3a negatively regulates acetylcholine receptor (AChR) clustering by repressing the expression of Rapsyn, an AChR-associated protein essential for AChR clustering. In cultured myotubes, treatment with Wnt3a or overexpression of beta-catenin, the condition mimicking the activation of the Wnt canonical pathway, inhibited Agrin-induced formation of AChR clusters. Moreover, Wnt3a treatment promoted dispersion of AChR clusters, and this effect was prevented by DKK1, an antagonist of the Wnt canonical pathway. Next, we investigated possible mechanisms underlying Wnt3a regulation of AChR clustering in cultured muscle cells. Interestingly, we found that Wnt3a treatment caused a decrease in the protein level of Rapsyn. In addition, Rapsyn promoter activity in cultured muscle cells was inhibited by the treatment with Wnt3a or beta-catenin overexpression. Forced expression of Rapsyn driven by a promoter that is not responsive to Wnt3a prevented the dispersing effect of Wnt3a on AChR clusters, suggesting that Wnt3a indeed acts to disperse AChR clusters by down-regulating the expression of Rapsyn. The role of Wnt/beta-catenin signaling in dispersing AChR clusters was also investigated in vivo by electroporation of Wnt3a or beta-catenin into mouse limb muscles, where ectopic Wnt3a or beta-catenin caused disassembly of postsynaptic apparatus. Together, these results suggest that Wnt/beta-catenin signaling plays a negative role for postsynaptic differentiation at the neuromuscular junction, probably by regulating the expression of synaptic proteins, such as Rapsyn.

Aberrant Development of Motor Axons and Neuromuscular Synapses inMyoD-NullMice

Myogenic regulatory factors (MRFs), muscle-specific transcription factors, are implicated in the activity-dependent regulation of nicotinic acetylcholine receptor (AChR) subunit genes. Here we show, with immunohistochemistry, Western blotting, and electron microscopy that MyoD, a member of the MRF family, also plays a role in fetal synapse formation. In the diaphragm of 14.5 d gestation (E14.5) wild-type and MyoD-/- mice, AChR clusters (the formation of which is under a muscle intrinsic program) are confined to a centrally located endplate zone. This distribution persists in wild-type adult muscles. However, beginning at E15.5 and extending to the adult, innervated AChR clusters are distributed all over the diaphragm of MyoD-/- mice, extending as far as the insertion of the diaphragm into the ribs. In wild-type muscle, motor axons terminate on clusters adjacent to the main intramuscular nerve; in MyoD-/- muscle, axonal bundles form extensive secondary branches that terminate on the widely distributed clusters. The number of AChR clusters on adult MyoD-/- and wild-type diaphram muscles is similar. Junctional fold density is reduced at MyoD-/- endplates, and the transition from the fetal (alpha, beta, gamma, delta) to adult-type (alpha, beta, delta, epsilon) AChRs is markedly delayed. However, MyoD-/- mice assemble a complex postsynaptic apparatus that includes muscle-specific kinase (MuSK), rapsyn, erbB, and utrophin.

Neural Agrin Activates a High-Affinity Receptor in C2 Muscle Cells that Is Unresponsive to Muscle Agrin

During synaptogenesis, agrin, released by motor nerves, causes the clustering of acetylcholine receptors (AChRs) in the skeletal muscle membrane. Although muscle alpha-dystroglycan has been postulated to be the receptor for the activity of agrin, previous experiments have revealed a discrepancy between the biological activity of soluble fragments of two isoforms of agrin produced by nerves and muscles, respectively, and their ability to bind alpha-dystroglycan. We have determined the specificity of the signaling receptor by investigating whether muscle agrin can block the activity of neural agrin on intact C2 myotubes. We find that a large excess of muscle agrin failed to inhibit either the number of AChR clusters or the phosphorylation of the AChR induced by picomolar concentrations of neural agrin. These results indicate that neural, but not muscle, agrin interacts with the signaling receptor. Muscle agrin did block the binding of neural agrin to isolated alpha-dystroglycan, however, suggesting either that alpha-dystroglycan is not the signaling receptor or that its properties in the membrane are altered. Direct assay of the binding of muscle or neural agrin to intact myotubes revealed only low-affinity binding. We conclude that the signaling receptor for agrin is a high-affinity receptor that is highly specific for the neural form.

The distribution of intracellular acetylcholine receptors and nuclei in avian slow muscle fibres during establishment of distributed synapses

The distribution of intracellular acetylcholine receptor was studied by 125I-alpha-bungarotoxin autoradiography as a measure of the local acetylcholine receptor synthesis at junctional and extrajunctional sites in single fibres of the developing anterior latissimus dorsi muscle of the chicken. Large (longer than 2 microns) acetylcholine receptor clusters characteristic of synaptic contacts were localized by immunofluorescence with anti-acetylcholine receptor antibodies. The distance between acetylcholine receptor clusters at embryonic day 11 was 166 +/- 10.5 microns and this distance did not increase despite growth until after 4 days posthatch. The distance between acetylcholine receptor clusters subsequently increased proportionately with the increase in the length of fibres. Intracellular acetylcholine receptors were labelled with 125I-alpha-BGT after first blocking cell-surface acetylcholine receptor with unlabelled alpha-BGT, and treatment with saponin. Intracellular acetylcholine receptor represented about 5-15% of total cellular acetylcholine receptor. Cycloheximide experiments indicated that 80-90% of intracellular acetylcholine receptor examined represented newly synthesized acetylcholine receptor. The spatial distribution of this pool, studied by autoradiography, was determined in relation to the acetylcholine receptor clusters labelled with anti-acetylcholine receptor antibody. Between embryonic day 11 and posthatch day 14 there was a continual increase in intracellular acetylcholine receptor at both junctional and extrajunctional parts of the fibres, but with the greater increases occurring at the junctional regions. Peaks of intracellular acetylcholine receptor became associated with an increasing number of acetylcholine receptor clusters so that by posthatch day 14 there was an 80% correspondence. The accumulation of newly synthesized intracellular acetylcholine receptor under acetylcholine receptor clusters was not the result of the aggregation of nuclei at these sites, suggesting that a higher rate of acetylcholine receptor synthesis per nucleus develops at distributed synaptic sites on anterior latissimus dorsi fibres.

Acetylcholine receptor clustering and nuclear movement in muscle fibers in culture.

We have studied the formation of acetylcholine receptor (AChR) clusters and the behavior of myonuclei in rat and chick skeletal muscle cells grown in cell culture. These cells were treated with a factor derived from Torpedo electric extracellular matrix, which causes a large increase in their number of AChR clusters. We found that these clusters were located preferentially in membrane regions above myonuclei. This cluster-nucleus colocalization is explained by our finding that most of the nuclei near clusters remain relatively stationary, while most of those away from clusters are able to translocate throughout the myotube. In some cases, clusters clearly formed first, then nuclei migrated underneath and became immobilized. If clustered AChRs later dispersed, their associated nuclei resumed moving. These results suggest that AChR clustering initiates an extensive cytoskeletal rearrangement that causes the subcluster localization of organelles, potentially providing a stable source of newly synthesized AChRs for insertion into the cluster.

Differential responses of L5 and rat primary muscle cells to factors in rat brain extract

Crude brain extract (100,000 g supernate from newborn or fetal rat brain homogenate) was studied for its effects on the number and distribution of acetylcholine receptors (AChRs) on myotubes of the L5 cloned myogenic cell line and compared to that of rat primary cultures. Gamma counting, light autoradiography and scanning electron microscopic autoradiography were used. We found that the L5 cells responded to the brain extract with an increase in the average AChR site density (2–5-fold) and with an increase in AChR clustering. Clustering was manifested by both an increase in the number of AChR clusters and in the ratio of receptor site density within clusters relative to that between clusters. The increase in average AChR site density was shown to be due to an increase in the rate of AChR insertion into the surface membrane with little change in the rate of receptor degradation. As also previously reported, the rat myotubes had a similar clustering response but only a very slight (∼ 1.2-fold) increase in average AChR site density. The surface area of myotubes was also increased slightly (∼ 1.2–1.3-fold) by the brain extract. Autoradiography viewed by scanning EM was found to be very useful in illustrating the shape and distribution of the receptor clusters. After the brain extract was fractionated on Sephadex G-200, the fractions with greatest clustering activity could be separated from those causing predominantly an increase in receptor site density. Increased receptor site density was primarily produced by the low molecular weight fractions (< 12 kD), whereas the strongest (but not exclusive) effect on clustering was produced by the high molecular weight fractions ( >140 kD). Furthermore, the two cell types assayed had different sensitivity to the different factors. L5 cells responded to both the high and low molecular weight factors while rat primary cells are sensitive primarily to the high molecular weight factors.

Evolution of cholinergic proteins in developing slow and fast skeletal muscles in chick embryo.

1. The cholinergic differentiation of two phenotypically muscles of the chick, the slow multiply innervated anterior latissimus dorsi (a.l.d.) and the fast focally innervated posterior latissimus dorsi (p.l.d.), was investigated during embryonic life and after hatching using both autoradiographical and biochemical methods. 2. The contents in total protein and in acetylcholinesterase activity follow similar development patterns in both muscles, but, after the 15th day in ovo, the accumulation of choline acetyltransferase activity and of acetylcholine nicotinic receptor sites as determined by alpha-bungarotoxin binding occurs at a faster rate in a.l.d. than in p.l.d. 3. In muscle of the p.l.d., a rapid increase of the total number of acetylcholine receptor clusters takes place after the 11th day of embryonic life although some clusters could be observed on myofibres as soon as the 4th day in ovo. 4. The rate of degradation of cholinergic receptor sites in chick muscle is constant around 28 hr up to the 10th day after hatching; thus the different rates of accumulation of acetylcholine receptor in a.l.d. and p.l.d., respectively, after the 15th day of embryonic life must be due to different rates of receptor synthesis. 5. The role of muscle activity in the biochemical differentiation of the developing motor end-plate was investigated in chick embryos which had been paralysed by repeated injections into the yolk sac of a curare-like agent, Flaxedil (May & Baker). 6. The total content in acetylcholinesterase of both a.l.d. and p.l.d. muscles is not significantly modified by paralysis. However, the histochemical staining of end-plates for acetylcholinesterase as well as the heavy form of this enzyme (19 . 5 S) are consistently reduced after Flaxedil injection. 7. In muscles from Flaxedil-treated embryos, the total content in acetylcholine receptor sites as determined by alpha-bungarotoxin binding is higher than in those from control embryos, whereas the rate of degradation of these sites is not significantly altered. 8. The localization of the acetylcholine receptors under the motor nerve terminals is not prevented by blocking muscle activity at the postsynaptic level. Clusters of receptor are still present, and there is no significant change in the number and distribution of these clusters along the myofibres of a.l.d. and p.l.d. muscles. 9. These results are discussed with respect to motor end-plate formation in multiply and focally innervated embryo muscles, and in relation to the control of cholinergic proteins distribution and synthesis by muscle activity.