Author Report for: Mars T

Expression of patatin-like phospholipase domain containing protein 7 (PNPLA7), also known as neuropathy target esterase-related esterase (NRE), a lysophospholipase, increases with fasting and decreases with feeding in mouse skeletal muscle, indicating it is regulated by insulin, counterregulatory hormones, such as glucocorticoids and catecholamines, and/or nutrients. In cultured mouse adipocytes insulin reduces Pnpla7 expression, underscoring the possibility that insulin regulates PNPLA7 in skeletal muscle. The first aim of this study was to establish whether PNPLA7 is functionally expressed in cultured human skeletal muscle cells. The second aim was to determine whether PNPLA7 is regulated by insulin, glucocorticoids, cAMP/protein kinase A pathway, and/or glucose. Cultured human skeletal muscle cells expressed PNPLA7 mRNA and protein. Gene silencing of PNPLA7 in myoblasts reduced the phosphorylation of 70 kDa ribosomal protein S6 kinase and ribosomal protein S6 as well as the abundance of alpha1-subunit of Na(+),K(+)-ATPase and acetyl-CoA carboxylase, indirectly suggesting that PNPLA7 is functionally important. In myotubes, insulin suppressed PNPLA7 mRNA at 1 g/L glucose, but not at low (0.5 g/L) or high (4.5 g/L) concentrations. Treatment with synthetic glucocorticoid dexamethasone and activator of adenylyl cyclase forskolin had no effect on PNPLA7 regardless of glucose concentration, while dibutyryl-cAMP, a cell-permeable cAMP analogue, suppressed PNPLA7 mRNA at 4.5 g/L glucose. The abundance of PNPLA7 protein correlated inversely with the glucose concentrations. Collectively, our results highlight that PNPLA7 in human myotubes is regulated by metabolic signals, implicating a role for PNPLA7 in skeletal muscle energy metabolism.

Acetylcholinesterase (AChE) and agrin, a heparan-sulfate proteoglycan, reside in the basal lamina of the neuromuscular junction (NMJ) and play key roles in cholinergic transmission and synaptogenesis. Unlike most NMJ components, AChE and agrin are expressed in skeletal muscle and alpha-motor neurons. AChE and agrin are also expressed in various other types of cells, where they have important alternative functions that are not related to their classical roles in NMJ. In this review, we first focus on co-cultures of embryonic rat spinal cord explants with human skeletal muscle cells as an experimental model to study functional innervation in vitro. We describe how this heterologous rat-human model, which enables experimentation on highly developed contracting human myotubes, offers unique opportunities for AChE and agrin research. We then highlight innovative approaches that were used to address salient questions regarding expression and alternative functions of AChE and agrin in developing human skeletal muscle. Results obtained in co-cultures are compared with those obtained in other models in the context of general advances in the field of AChE and agrin neurobiology.

Proteins in living organisms have names that are usually derived from their function in the biochemical system their discoverer was investigating. Typical examples are acetylcholinesterase and agrin; however, for both of these, various other functions that are not related to the cholinergic system have been revealed. Our investigations have been focused on the alternative roles of acetylcholinesterase and agrin in the processes of muscle development and regeneration. Previously, we described a role for agrin in the development of excitability in muscle contraction. In this study, we report the effects of agrin on secretion of interleukin 6 in developing human muscle. At the myoblast stage, agrin increases interleukin 6 secretion. This effect seems to be general as it was observed in all of the cell models analysed (human, mouse, cell lines). After fusion of myoblasts into myotubes, the effects of agrin are no longer evident, although agrin has further effects at the innervation stage, at least in in vitro innervated human muscle. These effects of agrin are another demonstration of its non-synaptic roles that are apparently developmental-stage specific. Our data support the view that acetylcholinesterase and agrin participate in various processes during development of skeletal muscle.

Organophosphorus compounds (OPs) and oximes may interfere with other molecules than AChE in the living systems, affecting in this way various cellular processes and underlying mechanisms. These non-cholinergic effects may contribute to the clinical status in OP poisoning and therefore deserve equal scientific attention. Here, we investigated the effects of tabun and oxime K048 on the processes known to be involved in muscle response to the environmental factors, like IL-6 release and the regulation of the heat shock proteins (HSPs). While IL-6 stimulates muscle regeneration, which follows well known OP-induced myopathy, HSPs have cytoprotective effect against various stress factors including xenobiotics. All our experiments were carried out on cultured human myoblasts, as the precursors of muscle regeneration. We found unchanged AChE mRNA level after tabun/K048 treatment meaning that tabun and K048 did not interfere with the transcription or stability of this mRNA in the time period tested, even if AChE catalytic activity was significantly affected. On the other hand, after myoblast exposure to tabun, we observed significant changes in the protein levels of HSP 27 and in the secretion of IL-6. Namely, secretion of IL-6 decreased to 53% and the level of HSP 27 increased by 34% compared to the control level. Both effects were attenuated if myoblasts were pretreated with oxime K048, but not if they were treated with K048 after exposure to tabun. The molecular mechanism underlying these effects remains to be elucidated. However, it seems that these effects could be associated with OPs and oximes as a specific group of compounds rather than as a specific compound itself. Overall, the effects of OPs and oximes demonstrated here might play an important role in muscle regeneration which importantly determines the final outcome of OP myotoxicity.

Acetylcholinesterase (AChE) and agrin play unique functional roles in the neuromuscular junction (NMJ). AChE is a cholinergic and agrin a synaptogenetic component. In spite of their different functions, they share several common features: their targeting is determined by alternative splicing; unlike most other NMJ components they are expressed in both, muscle and motor neuron and both reside on the synaptic basal lamina of the NMJ. Also, both were reported to play various nonjunctional roles. However, while the origin of basal lamina bound agrin is undoubtedly neural, the neural origin of AChE, which is anchored to the basal lamina with collagenic tail ColQ, is elusive. Hypothesizing that motor neuron proteins targeted to the NMJ basal lamina share common temporal pattern of expression, which is coordinated with the formation of basal lamina, we compared expression of agrin isoforms with the expression of AChE-T and ColQ in the developing rat spinal cord at the stages before and after the formation of NMJ basal lamina. Cellular origin of AChE-T and agrin was determined by in situ hybridization and their quantitative levels by RT PCR. We found parallel increase in expression of the synaptogenetic (agrin 8) isoform of agrin and ColQ after the formation of basal lamina supporting the view that ColQ bound AChE and agrin 8 isoform are destined to the basal lamina. Catalytic AChE-T subunit and agrin isoforms 19 and 0 followed different expression patterns. In accordance with the reports of other authors, our investigations also revealed various alternative functions for AChE and agrin. We have already demonstrated participation of AChE in myoblast apoptosis; here we present the evidence that agrin promotes the maturation of heavy myosin chains and the excitation-contraction coupling. These results show that common features of AChE and agrin extend to their capacity to play multiple roles in muscle development.

The best established role of acetylcholinesterase (EC 3.1.1.7, AChE) is termination of neurotransmission at cholinergic synapses. However, AChE is also located at sites, where no other cholinergic components are present and there is accumulating evidence for non-cholinergic functions of this protein. In the process of skeletal muscle formation, AChE is expressed already at the stage of mononuclear myoblast, which is long before other cholinergic components can be demonstrated in this tissue. Myoblast proliferation is an essential step in muscle regeneration and is always accompanied by apoptosis. Since there are several reports demonstrating AChE participation in apoptosis one can hypothesize that early AChE expression in myoblasts reflects the development of the apoptotic apparatus in these cells. Here we tested this hypothesis by following the effect of siRNA AChE silencing on apoptotic markers and by determination of AChE level after staurosporine-induced apoptosis in cultured human myoblasts. Decreased apoptosis in siRNA AChE silenced myoblasts and increased AChE expression in staurosporine-treated myoblasts confirmed AChE involvement in apoptosis. The three AChE splice variants were differently affected by staurosporine-induced apoptosis. The hydrophobic (H) variant appeared unaffected, a tendency towards increase of tailed (T) variant was detected, however the highest, 8-fold increase was observed for readthrough (R) variant. In the light of these findings AChE appears to be a potential therapeutic target at muscle injuries including organophosphate myopathy.

Acetylcholinesterase (EC 3.1.1.7, AChE) is one of the components of the neuromuscular junction (NMJ). Its expression and targeting in the skeletal muscle fiber is therefore under the control of the mechanisms responsible for the formation of the highly complex structure of this synapse. Recently, it has been demonstrated that myotubes of the C2C12 mouse muscle cell line form highly differentiated pretzel-like postsynaptic accumulations of acetylcholine receptors (AChRs) in the complete absence of the nerve if they are cultured on the laminin coating. This finding questions previously stressed importance of the nerve-derived factors in NMJ synaptogenesis and therefore deserves additional testing. The aim of this paper was to test whether the reported nerve-independency can be demonstrated also in the cultured human muscle meaning that the findings on C2C12 cultures can be extrapolated also to the human muscle. In our experiments aneurally cultured human myotubes failed to form AChR clusters on its surface, no matter if they were grown on normal gelatine or laminin coating. However, when innervated by neurons extending from the rat embryonic spinal cord, human myotubes formed AChR clusters with elaborate topography but strictly on the areas contacted by the nerve. One can hypothesize that higher nerve dependency of the NMJ synaptogenesis in humans in comparison to other species reflects species-specific differences in the organization of movement. Humans have the highest "fractionation of movement" capacity which probably requests different, more nerve-controlled development of the motor system including nerve-restricted development of the neuromuscular contacts.

In spite of several reports demonstrating that acetylcholinesterase (AChE [EC 3.1.1.7]) expression is importantly regulated at the level of its mRNA, we still know little about the relationship between AChE mRNA level and the level of mature, catalytically active enzyme in the cell. Better insight into this relationship is, however, essential for our understanding of the molecular pathways underlying AChE synthesis in living cells. We have approached this problem previously (Grubic et al., 1995; Brank et al., 1998; Mis et al., 2003; Jevsek et al., 2004); however, recently introduced small interfering RNA (siRNA) methodology, which allows blockade of gene expression at the mRNA level, opens new possibilities in approaching the AChE mRNA-AChE activity relationship. With this technique one can eliminate AChE mRNA in the cell, specifically and at selected times, and follow the effects of such treatment at the mature enzyme level. In this study we followed AChE activity in siRNA-treated cultured human myoblasts. Our aim was to find out how the temporal profile of the AChE mRNA decrease is reflected at the level of AChE activity under normal conditions and after inhibition of preexisting AChE by diisopropyl phosphorofluoridate (DFP).AChE activity was determined at selected time intervals after siRNA treatment in both myoblast homogenates and in culture medium to follow the effects of siRNA treatment at the level of intracellular AChE synthesis and at the level of AChE secreted from the cell.

The results of our recent investigations on the expression and distribution of acetylcholinesterase (EC. 3.1.1.7, AChE) in the experimental model of the in vitro innervated human muscle are summarized and discussed here. This is the only model allowing studies on AChE expression at all stages of the neuromuscular junction (NMJ) formation in the human muscle. Since it consists not only of the motor neurons and myotubes but also of glial cells, which are essential for the normal development of the motor neurons, NMJs become functional and differentiated in this system. We followed AChE expression at various stages of the NMJ formation and in the context of other events characteristic for this process. Neuronal and muscular part were analysed at both, mRNA and mature enzyme level. AChE is expressed in motor neurons and skeletal muscle at the earliest stages of their development, long before NMJ starts to form and AChE begins to act as a cholinergic component. Temporal pattern of AChE mRNA expression in motor neurons is similar to the pattern of mRNA encoding synaptogenetic variant of agrin. There are no AChE accummulations at the NMJ at the early stage of its formation, when immature clusters of nicotinic receptors are formed at the neuromuscular contacts and when occasional NMJ-mediated contractions are already observed. The transformation from immature, bouton-like neuromuscular contacts into differentiated NMJs with mature, compact receptor clusters, myonuclear accumulations and dense AChE patches begins at the time when basal lamina starts to form in the synaptic cleft. Our observations support the concept that basal lamina formation is the essential event in the transformation of immature neuromuscular contact into differentiated NMJ, with the accumulation of not only muscular but also neuronal AChE in the synaptic cleft.

Synaptic basal lamina is interposed between the pre- and postsynaptic membrane of the neuromuscular junction (NMJ). This position permits deposition of basal lamina-bound NMJ components of both neuronal and muscle fibre origin. One such molecule is acetylcholinesterase (AChE). The origin of NMJ AChE has been investigated previously as the answer would elucidate the relative contributions of muscle fibers and motor neurons to NMJ formation. However, in the experimental models used in prior investigations either the neuronal or muscular components of the NMJs were removed, or the NMJs were poorly differentiated. Therefore, the question of AChE origin in the intact and functional NMJ remains open. Here, we have approached this question using an in vitro model in which motor neurons, growing from embryonic rat spinal cord explants, form well differentiated NMJs with cultured human myotubes. By immunocytochemical staining with species-specific anti-AChE antibodies, we are able to differentiate between human (muscular) and rat (neuronal) AChE at the NMJ. We observed strong signal at the NMJ after staining with human AChE antibodies, which suggests a significant muscular AChE contribution. However, a weaker, but still clearly recognizable signal is observed after staining with rat AChE antibodies, suggesting a smaller fraction of AChE was derived from motor neurons. This is the first report demonstrating that both motor neuron and myotube contribute synaptic AChE under conditions where they interact with each other in the formation of an intact and functional NMJ.

In spite of intensive investigations, the roles of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8) in the central nervous system (CNS) remain unclear. A role recently proposed for BuChE as an explanation for survival of AChE knockout mice is compensation for AChE activity if it becomes insufficient. Neuronal contribution of both enzymes to the cholinesterase pool in the neuromuscular junction has also been suggested. These proposals imply that BuChE expression follows that of AChE and that, in addition to AChE, BuChE is also expressed in alpha-motor neurons. However, these assumptions have not yet been properly tested. Histochemical approaches to these problems have been hampered by a number of problems that prevent unambiguous interpretation of results. In situ hybridization (ISH) of mRNAs encoding AChE and BuChE, which is the state-of-the-art approach, has not yet been done. Here we describe rapid nonradioactive ISH for the localization of mRNAs encoding AChE and BuChE. Various probes and experimental conditions had been tested to obtain reliable localization. In combination with RT-PCR, ISH revealed that, in rat spinal cord, cells expressing AChE mRNA also express BuChE mRNA but in smaller quantities. alpha-Motor neurons had the highest levels of both mRNAs. Virtual absence of transcripts encoding AChE and BuChE in glia might reflect a discrepancy between mRNA and enzyme levels previously reported for cholinesterases.

Protein expression can be controled at different levels. Understanding acetylcholinesterase (EC. 3.1.1.7, AChE) expression in the living organisms therefore necessitates: (1) determination and mapping of control levels of AChE metabolism; (2) identification of the regulatory factors acting at these levels; and (3) detailed insight into the mechanisms of action of these factors. Here we summarize the results of our studies on the regulation of AChE expression in the mammalian skeletal muscle. Three experimental models were employed: in vitro innervated human muscle, mechanically denervated adult fast rat muscle, and the glucocorticoid treated fast rat muscle. In situ hybridization of AChE mRNA, combined with AChE histochemistry, revealed that different distribution patterns of AChE, observed during in vitro ontogenesis and synaptogenesis of human skeletal muscle, reflect alterations in the distribution of AChE mRNA (Z. Grubic, R. Komel, W.F. Walker, A.F. Miranda, Myoblast fusion and innervation with rat motor nerve alter the distribution of acetylcholinesterase and its mRNA in human muscle cultures, Neuron 14 (1995) 317-327). To study the mechanisms of AChE mRNA loss in denervated adult rat skeletal muscle, we exposed deproteinated AChE mRNA to various subcellular fractions in vitro. Fractions were isolated from the normal and denervated rat sternomastoideus muscle. We found significantly increased, but non-specific AChE mRNA degradation capacities in the three fractions studied, suggesting that increased susceptibility of muscle mRNA to degradation might be at least partly responsible for the decreased AChE mRNA observed under such conditions (K. Zajc-Kreft, S. Kreft, Z. Grubic, Degradation of AChE mRNA in the normal and denervated rat skeletal muscle, Book of Abstracts, The Sixth International Meeting on Cholinesterases, La Jolla, CA, March 20-24, 1998, p. A3.). In adult fast rat muscle, treated chronically with glucocorticoids, we found the fraction of early synthesized AChE molecular forms to be reduced and AChE mRNA unchanged. This observation is consistent with the explanation that translation and/or early post-translational processes are impaired under such conditions (M. Brank, K. Zajc-Kreft, S. Kreft, R. Komel, Z. Grubic, Biogenesis of acetylcholinesterase is impaired, although its mRNA level remains normal, in the glucocorticoid-treated rat skeletal muscle, Eur. J. Biochem. 251 (1998) 374-381). The AChE mRNA level is therefore important but not the only control level of AChE expression in the mammalian skeletal muscle.

Unlike the majority of mammalian tissues, which have mononuclear cells as a basic unit of the tissue composition, skeletal muscle fiber is a polynuclear syncytium. Polynuclear organization offers an additional possibility for the regulation of protein expression: it can also be controlled at the level of myonuclear distribution and specialization. Distribution of myonuclei can be considered as the distribution of genes. Variability in gene concentration may have important impact on the regional differentiation of the muscle fiber, since it leads to different regional concentrations of various gene products including factors controlling their expression. The aim of the present study was: 1) to provide morphometrical data on the myonuclear distribution in the junctional and extrajunctional regions of the normal fast rat muscle fiber, and 2) to analyze, whether morphometrical parameters of nuclear distribution change after mechanical denervation of this muscle. Single muscle fibers were isolated from the normal and denervated fast rat m. sternomastoideus. Their neuromuscular junctions were stained by thiocholine histochemical procedure and myonuclei were fluorescently labeled by Hoechst 33342. Myonuclear distribution in each individual muscle fiber was morphometrically analyzed on the image analyzer. Synaptic concentrations of myonuclei were found to exceed extrasynaptic concentrations by a factor of 17. The number of myonuclei accumulated at the endplates did not change after one or two weeks of denervation, neither did the morphometric parameters of these nuclei. A higher concentration of myonuclei due to muscle atrophy was observed in the extrasynaptic region and the longitudinal axis of these nuclei also became significantly shorter. Unchanged morphometric parameters of the junctional myonuclei after denervation are indicative of either irreversibility of the nerve-induced formation of nuclear clusters in this region or persistence of the factors responsible for their formation and maintenance.