The NMJ as a model synapse: New perspectives on formation, synaptic transmission and maintenance: Acetylcholinesterase at the neuromuscular junction.

Acetylcholinesterase (AChE) is an essential enzymatic component of the neuromuscular junction where it is responsible for terminating neurotransmission by the cholinergic motor neurons. The enzyme at the neuromuscular junction (NMJ) is contributed primarily by the skeletal muscle where it is produced at higher levels in the post-synaptic region of the fibers. The major form of AChE at the NMJ is a large asymmetric form consisting of three tetramers covalently attached to a three-stranded collagen-like tail which is responsible for anchoring it to the synaptic basal lamina. Its location and expression is regulated to a large extent by the motor neurons and occurs at the transcriptional, translational and post-translational levels. While its expression can be quite rapid in tissue cultured cells, its half-life in vivo appears to be quite long, about three weeks, although more rapidly turning over pools have been described. Finally the essential nature of this enzyme is underscored by the fact that no naturally occurring null mutations of the catalytic subunit have been described in higher organisms and the few dozen humans carrying mutations in the collagen tail responsible for anchoring the enzyme at the NMJ are severely affected.

Overexpression of PGC-1α in aging muscle enhances a subset of young-like molecular patterns.

PGC-1α is a transcriptional co-activator known as the master regulator of mitochondrial biogenesis. Its control of metabolism has been suggested to exert critical influence in the aging process. We have aged mice overexpressing PGC-1α in skeletal muscle to determine whether the transcriptional changes reflected a pattern of expression observed in younger muscle. Analyses of muscle proteins showed that Pax7 and several autophagy markers were increased. In general, the steady-state levels of several muscle proteins resembled that of muscle from young mice. Age-related mtDNA deletion levels were not increased by the PGC-1α-associated increase in mitochondrial biogenesis. Accordingly, age-related changes in the neuromuscular junction were minimized by PGC-1α overexpression. RNA-Seq showed that several genes overexpressed in the aged PGC-1α transgenic are expressed at higher levels in young when compared to aged skeletal muscle. As expected, there was increased expression of genes associated with energy metabolism but also of pathways associated with muscle integrity and regeneration. We also found that PGC-1α overexpression had a mild but significant effect on longevity. Taken together, overexpression of PGC-1α in aged muscle led to molecular changes that resemble the patterns observed in skeletal muscle from younger mice.

Biogenesis, assembly and trafficking of acetylcholinesterase.

Acetylcholinesterase (AChE) is expressed as several homomeric and heterooligomeric forms in a wide variety of tissues such as neurons in the central and peripheral nervous systems and their targets including skeletal muscle, endocrine and exocrine glands. In addition, glycolipid-anchored forms are expressed in erythropoietic and lymphopoietic cells. While transcriptional and post-transcriptional regulation is important for determining which AChE oligomeric forms are expressed in a given tissue, translational and post-translational regulatory mechanisms at the level of protein folding, assembly and sorting play equally important roles in assuring that the AChE molecules reach their intended sites on the cell surface in the appropriate numbers. This brief review will focus on the latter events in the cell with the goal of providing novel therapeutic interventional strategies for the treatment of organophosphate and carbamate pesticide and nerve agent exposure. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

Rescue and Stabilization of Acetylcholinesterase in Skeletal Muscle by N-terminal Peptides Derived from the Noncatalytic Subunits.

The vast majority of newly synthesized acetylcholinesterase (AChE) molecules do not assemble into catalytically active oligomeric forms and are rapidly degraded intracellularly by the endoplasmic reticulum-associated protein degradation pathway. We have previously shown that AChE in skeletal muscle is regulated in part post-translationally by the availability of the noncatalytic subunit collagen Q, and others have shown that expression of a 17-amino acid N-terminal proline-rich attachment domain of collagen Q is sufficient to promote AChE tetramerization in cells producing AChE. In this study we show that muscle cells, or cell lines expressing AChE catalytic subunits, incubated with synthetic proline-rich attachment domain peptides containing the endoplasmic reticulum retrieval sequence KDEL take up and retrogradely transport them to the endoplasmic reticulum network where they induce assembly of AChE tetramers. The peptides act to enhance AChE folding thereby rescuing them from reticulum degradation. This enhanced folding efficiency occurs in the presence of inhibitors of protein synthesis and in turn increases total cell-associated AChE activity and active tetramer secretion. Pulse-chase studies of isotopically labeled AChE molecules show that the enzyme is rescued from intracellular degradation. These studies provide a mechanistic explanation for the large scale intracellular degradation of AChE previously observed and indicate that simple peptides alone can increase the production and secretion of this critical synaptic enzyme in muscle tissue.

Transient systemic mtDNA damage leads to muscle wasting by reducing the satellite cell pool.

With age, muscle mass and integrity are progressively lost leaving the elderly frail, weak and unable to independently care for themselves. Defined as sarcopenia, this age-related muscle atrophy appears to be multifactorial but its definite cause is still unknown. Mitochondrial dysfunction has been implicated in this process. Using a novel transgenic mouse model of mitochondrial DNA (mtDNA) double-strand breaks (DSBs) that presents a premature aging-like phenotype, we studied the role of mtDNA damage in muscle wasting. We caused DSBs in mtDNA of adult mice using a ubiquitously expressed mitochondrial-targeted endonuclease, mito-PstI. We found that a short, transient systemic mtDNA damage led to muscle wasting and a decline in locomotor activity later in life. We found a significant decline in muscle satellite cells, which decreases the muscle's capacity to regenerate and repair during aging. This phenotype was associated with impairment in acetylcholinesterase (AChE) activity and assembly at the neuromuscular junction (NMJ), also associated with muscle aging. Our data suggests that systemic mitochondrial dysfunction plays important roles in age-related muscle wasting by preferentially affecting the myosatellite cell pool.

Biglycan is an extracellular MuSK binding protein important for synapse stability.

The receptor tyrosine kinase MuSK is indispensable for nerve-muscle synapse formation and maintenance. MuSK is necessary for prepatterning of the endplate zone anlage and as a signaling receptor for agrin-mediated postsynaptic differentiation. MuSK-associated proteins such as Dok7, LRP4, and Wnt11r are involved in these early events in neuromuscular junction formation. However, the mechanisms regulating synapse stability are poorly understood. Here we examine a novel role for the extracellular matrix protein biglycan in synapse stability. Synaptic development in fetal and early postnatal biglycan null (bgn(-/o)) muscle is indistinguishable from wild-type controls. However, by 5 weeks after birth, nerve-muscle synapses in bgn(-/o) mice are abnormal as judged by the presence of perijunctional folds, increased segmentation, and focal misalignment of acetylcholinesterase and AChRs. These observations indicate that previously occupied presynaptic and postsynaptic territory has been vacated. Biglycan binds MuSK and the levels of this receptor tyrosine kinase are selectively reduced at bgn(-/o) synapses. In bgn(-/o) myotubes, the initial stages of agrin-induced MuSK phosphorylation and AChR clustering are normal, but the AChR clusters are unstable. This stability defect can be substantially rescued by the addition of purified biglycan. Together, these results indicate that biglycan is an extracellular ligand for MuSK that is important for synapse stability.

Translational regulation of acetylcholinesterase by the RNA-binding protein Pumilio-2 at the neuromuscular synapse.

Acetylcholinesterase (AChE) is highly expressed at sites of nerve-muscle contact where it is regulated at both the transcriptional and post-transcriptional levels. Our understanding of the molecular mechanisms underlying its regulation is incomplete, but they appear to involve both translational and post-translational events as well. Here, we show that Pumilio-2 (PUM2), an RNA binding translational repressor, is highly localized at the neuromuscular junction where AChE mRNA concentrates. Immunoprecipitation of muscle cell extracts with a PUM2 specific antibody precipitated AChE mRNA, suggesting that PUM2 binds to the AChE transcripts in a complex. Gel shift assays using a bacterially expressed PUM2 RNA binding domain showed specific binding using wild type AChE 3'-UTR RNA segment that was abrogated by mutation of the consensus recognition site. Transfecting skeletal muscle cells with shRNAs specific for PUM2 up-regulated AChE expression, whereas overexpression of PUM2 decreased AChE activity. We conclude that PUM2 binds to AChE mRNA and regulates AChE expression translationally at the neuromuscular synapse. Finally, we found that PUM2 is regulated by the motor nerve suggesting a trans-synaptic mechanism for locally regulating translation of specific proteins involved in modulating synaptic transmission, analogous to CNS synapses.

Increased muscle PGC-1alpha expression protects from sarcopenia and metabolic disease during aging.

Aging is a major risk factor for metabolic disease and loss of skeletal muscle mass and strength, a condition known as sarcopenia. Both conditions present a major health burden to the elderly population. Here, we analyzed the effect of mildly increased PGC-1alpha expression in skeletal muscle during aging. We found that transgenic MCK-PGC-1alpha animals had preserved mitochondrial function, neuromuscular junctions, and muscle integrity during aging. Increased PGC-1alpha levels in skeletal muscle prevented muscle wasting by reducing apoptosis, autophagy, and proteasome degradation. The preservation of muscle integrity and function in MCK-PGC-1alpha animals resulted in significantly improved whole-body health; both the loss of bone mineral density and the increase of systemic chronic inflammation, observed during normal aging, were prevented. Importantly, MCK-PGC-1alpha animals also showed improved metabolic responses as evident by increased insulin sensitivity and insulin signaling in aged mice. Our results highlight the importance of intact muscle function and metabolism for whole-body homeostasis and indicate that modulation of PGC-1alpha levels in skeletal muscle presents an avenue for the prevention and treatment of a group of age-related disorders.

Limiting role of protein disulfide isomerase in the expression of collagen-tailed acetylcholinesterase forms in muscle.

The expression of acetylcholinesterase (AChE) in skeletal muscle is regulated by muscle activity; however, the underlying molecular mechanisms are incompletely understood. We show here that the expression of the synaptic collagen-tailed AChE form (ColQ-AChE) in quail muscle cultures can be regulated by muscle activity post-translationally. Inhibition of thiol oxidoreductase activity decreases expression of all active AChE forms. Likewise, primary quail myotubes transfected with protein disulfide isomerase (PDI) short hairpin RNAs showed a significant decrease of both the intracellular pool of all collagen-tailed AChE forms and cell surface AChE clusters. Conversely, overexpression of PDI, endoplasmic reticulum protein 72, or calnexin in muscle cells enhanced expression of all collagen-tailed AChE forms. Overexpression of PDI had the most dramatic effect with a 100% increase in the intracellular ColQ-AChE pool and cell surface enzyme activity. Moreover, the levels of PDI are regulated by muscle activity and correlate with the levels of ColQ-AChE and AChE tetramers. Finally, we demonstrate that PDI interacts directly with AChE intracellularly. These results show that collagen-tailed AChE form levels induced by muscle activity can be regulated by molecular chaperones and suggest that newly synthesized exportable proteins may compete for chaperone assistance during the folding process.

Dissociation of transcription, translation, and assembly of collagen-tailed acetylcholinesterase in skeletal muscle.

The synaptic form of acetylcholinesterase (AChE) in skeletal muscle ColQ-AChE derives from two separate genes encoding the catalytic and the non-catalytic collagenic tail (ColQ) subunits, respectively. ColQ-AChE expression is regulated by muscle activity; however, how this regulation takes place in skeletal muscle remains poorly understood. In this study, we overexpressed or knocked down ColQ expression in skeletal muscle and found that the level of this non-catalytic component by itself was sufficient to change the levels of total AChE activity by promoting assembly of higher order oligomeric forms including the collagen-tailed forms. These results initially suggested that ColQ could be limiting in the assembly of synaptic ColQ-AChE during development and differentiation. We then determined the levels of ColQ protein and ColQ mRNA during primary quail muscle cell development and differentiation in culture (QMCs) and as a function of muscle activity. Surprisingly, we found dissociation between transcription and translation of the non-catalytic subunit from its assembly into ColQ-AChE. Furthermore, we found that the vast majority of the steady state ColQ molecules in mature quail muscle cultures are not assembled into ColQ-AChE, suggesting that they are either rapidly degraded or have alternative function(s).

Targeting acetylcholinesterase to the neuromuscular synapse.

The collagen-tailed form of acetylcholinesterase (ColQ-AChE) is the major if not unique form of the enzyme associated with the specialized synaptic basal lamina at the neuromuscular junction (NMJ). This enzyme form consists of both catalytic and non-catalytic subunits encoded by separate genes, assembled as three enzymatic tetramers attached to the three-stranded collagen-like tail. We have previously shown that catalytic subunits are assembled in the rough endoplasmic reticulum and that after approximately 90min a subset of these tetramers assemble with collagenic tail subunits in the Golgi apparatus. In muscle, blocking ER to Golgi transport with Brefeldin A prevents the appearance of ColQ-AChE, consistent with assembly of asymmetric forms in the Golgi. Moreover, newly synthesized and assembled ColQ-AChE associates with perlecan intracellularly and can only be co-immunoprecipitated with anti-perlecan antibodies 90min after the first appearance of catalytic subunits. Once assembled, the ColQ-AChE/perlecan complex is externalized where it co-localizes with other components of the NMJ including dystroglycan, rapsyn, laminin and MuSK. These clusters tend to form over the nuclei that are expressing the components, suggesting local vectorial transport to the cell surface, and may form a primary scaffold that in turn can capture other molecular constituents of the neuromuscular synapse. While most AChE clusters on quail myotubes are devoid of acetylcholine receptors, treatment of the culture with recombinant agrin results in a rapid translocation of receptors to the AChE clusters in less than 4h. It remains to be determined if MuSK is localized to the clusters. In vivo, AChE transcripts and enzyme are more highly expressed at the NMJs, implying higher rates of AChE translation and assembly in the synaptic regions, and hence more ColQ-AChE for localized export. We have previously shown that binding sites for ColQ-AChE are concentrated at sites of nerve-muscle contact where they colocalize with AChR and perlecan. ColQ-AChE binds directly to perlecan using solid phase microtiter plate assay, the Biacore assay, and co-immunoprecipitations. Moreover, perlecan binds to dystroglycan at the NMJ. In perlecan or dystroglycan null mice there is no accumulation of AChE at the NMJ, supporting the hypothesis that this heparan sulfate proteoglycan is an essential component of the ColQ-AChE localization mechanism. Together, these studies suggest a model of synaptic development whereby AChE can be targeted to and clustered on the muscle membrane together with dystroglycan and perlecan to form scaffolds to which AChR can be clustered through activation of the MuSK receptor. At mature synapses ColQ-AChE is secreted directly into the synaptic cleft where it binds to the heparan sulfate proteoglycan perlecan as well as potentially other molecules including MuSK, as was recently reported.

C-terminal and heparin-binding domains of collagenic tail subunit are both essential for anchoring acetylcholinesterase at the synapse.

The collagen-tailed form of acetylcholinesterase (A(12)-AChE) appears to be localized at the neuromuscular junction in association with the transmembrane dystroglycan complex through binding of its collagenic tail (ColQ) to the proteoglycan perlecan. The heparan sulfate binding domains (HSBD) of ColQ are thought to be involved in anchoring ColQ to the synaptic basal lamina. The C-terminal domain (CTD) of ColQ is also likely involved, but there has been no direct evidence. Mutations in COLQ cause endplate AChE deficiency in humans. Nine previously reported and three novel mutations are in CTD of ColQ, and most CTD mutations do not abrogate formation of A(12)-AChE in transfected COS cells. Patient endplates, however, are devoid of AChE, suggesting that CTD mutations affect anchoring of ColQ to the synaptic basal lamina. Based on our observations that purified AChE can be transplanted to the heterologous frog neuromuscular junction, we tested insertion competence of nine naturally occurring CTD mutants and two artificial HSBD mutants. Wild-type human A(12)-AChE inserted into the frog neuromuscular junction, whereas six CTD mutants and two HSBD mutants did not. Our studies establish that the CTD mutations indeed compromise anchoring of ColQ and that both HSBD and CTD are essential for anchoring ColQ to the synaptic basal lamina.

Localization of the calcitonin gene-related peptide receptor complex at the vertebrate neuromuscular junction and its role in regulating acetylcholinesterase expression.

The calcitonin gene-related peptide (CGRP) is released by motor neurons where it exerts both short and long term effects on skeletal muscle fibers. In addition, sensory neurons release CGRP on the surrounding vasculature where it is in part responsible for local vasodilation following muscle contraction. Although CGRP-binding sites have been demonstrated in whole muscle tissue, the type of CGRP receptor and its associated proteins or its cellular localization within the tissue have not been described. Here we show that the CGRP-binding protein referred to as the calcitonin receptor-like receptor is highly concentrated at the avian neuromuscular junction together with its two accessory proteins, receptor activity modifying protein 1 and CGRP-receptor component protein, required for ligand specificity and signal transduction. Using tissue-cultured skeletal muscle we show that CGRP stimulates an increase in intracellular cAMP that in turn initiates down-regulation of acetylcholinesterase expression at the transcriptional level, and, more specifically, inhibits expression of the synaptically localized collagen-tailed form of the enzyme. Together, these studies suggest a specific role for CGRP released by spinal cord motoneurons in modulating synaptic transmission at the neuromuscular junction by locally inhibiting the expression of acetylcholinesterase, the enzyme responsible for terminating acetylcholine neurotransmission.

Expression and localization of acetylcholinesterase at the neuromuscular junction.

Acetylcholinesterase (AChE) was the first identified synaptic component, characterized by its catalytic affinity for choline esters, and for a long time provided the conceptual scaffold for studies of the neuromuscular junction (NMJ). AChE was the first protein localized to the NMJ, in the 1930s, and its local expression shown to be regulated by the motoneuron in 1937. By the late 1940s a histochemical procedure was developed to visualize the enzyme at synapses, opening up a new era in the study of synaptic structure and function. This enzyme was also the first synaptic component to be purified, quantified, and later crystallized for structural and functional studies. Thus, historically, AChE has played a pivotal role in our understanding of NMJ development and function.AChE occurs in multiple oligomeric forms differing in their hydrophobic character and association with non-catalytic subunits that act as targeting sequences to insure correct localization at the cellular level. The predominant, if not unique, form of the enzyme responsible for terminating neuromuscular transmission is the collagen-tailed form of the enzyme attached to the synaptic basal lamina. This very large protein consists of three catalytic tetramers covalently linked to the three-stranded collagen-like tail (ColQ). The transcripts encoding AChE in muscle are preferentially expressed at sites of nerve-muscle contact and the AChE locally synthesized and assembled. Once externalized, the enzyme appears to be localized through interactions with the proteoglycan perlecan and subsequently covalently attached to the synaptic extracellular matrix. Thus a hierarchical series of events involving localized transcription and translation of the catalytic and non-catalytic subunits, localized assembly and secretion, and finally localized attachment to elements of the extracellular matrix insures that the enzyme is targeted to the correct location.

Absence of acetylcholinesterase at the neuromuscular junctions of perlecan-null mice.

The collagen-tailed form of acetylcholinesterase (AChE) is concentrated at the vertebrate neuromuscular junction (NMJ), where it is responsible for rapidly terminating neurotransmission. This unique oligomeric form of AChE, consisting of three tetramers covalently attached to a collagen-like tail, is more highly expressed in innervated regions of skeletal muscle fibers, where it is externalized and attached to the synaptic basal lamina interposed between the nerve terminal and the receptor-rich postsynaptic membrane. Although it is clear that the enzyme is preferentially synthesized in regions of muscle contacted by the motoneuron, the molecular events underlying its localization to the NMJ are not known. Here we show that perlecan, a multifunctional heparan sulfate proteoglycan concentrated at the NMJ, is the unique acceptor molecule for collagen-tailed AChE at sites of nerve-muscle contact and is the principal mechanism for localizing AChE to the synaptic basal lamina.