Biomedical subjects

Marko Jevsek

Publications and source records attributed to Marko Jevsek.

9 recordsLinked to original sources

PubMed2006

High dexamethasone concentration prevents stimulatory effects of TNF-alpha and LPS on IL-6 secretion from the precursors of human muscle regeneration.

A frequent finding in patients surviving critical illness myopathy is chronic muscle dysfunction. Its pathogenesis is mostly unknown; one explanation could be that muscle regeneration, which normally follows myopathy, is insufficient in these patients because of a high glucocorticoid level in their blood. Glucocorticoids can prevent stimulatory effects of proinflammatory factors on the interleukin (IL)-6 secretion, diminishing in this way the autocrine and paracrine IL-6 actions known to stimulate proliferation at the earliest, myoblast stage of muscle formation. To test this hypothesis, we compared the effects of major proinflammatory agents [tumor necrosis factor (TNF)-alpha and endotoxin lipopolysaccharide (LPS)] on the IL-6 secretion from the muscle precursors and then studied the influence of dexamethasone (Dex) on these effects. Mononuclear myoblasts, which still proliferate, were compared with myotubes in which this capacity is already lost. For correct interpretation of results, cultures were examined for putative apoptosis and necrosis. We found that constitutive secretion of IL-6 did not differ significantly between myoblasts and myotubes; however, the TNF-alpha- and LPS-stimulated IL-6 release was more pronounced (P < 0.001) in myoblasts. Dex, applied at the 0.1-100 nM concentration range, prevented constitutive and TNF-alpha- and LPS-stimulated IL-6 release at both developmental stages but only at high concentration (P < 0.01). Although there are still missing links to it, our results support the concept that high concentrations of glucocorticoids, met in critically ill patients, prevent TNF-alpha- and LPS-stimulated IL-6 secretion. This results in reduced IL-6-mediated myoblast proliferation, leading to the reduced final mass of the regenerated muscle.

Cell Differentiation↗

PubMed2006

CD24 is expressed by myofiber synaptic nuclei and regulates synaptic transmission.

The genes encoding several synaptic proteins, including acetylcholine receptors, acetylcholinesterase, and the muscle-specific kinase, MuSK, are expressed selectively by a small number of myofiber nuclei positioned near the synaptic site. Genetic analysis of mutant mice suggests that additional genes, expressed selectively by synaptic nuclei, might encode muscle-derived retrograde signals that regulate the differentiation of motor axon terminals. To identify candidate retrograde signals, we used a microarray screen to identify genes that are preferentially expressed in the synaptic region of muscle, and we analyzed one such gene, CD24, further. We show that CD24, which encodes a small, variably and highly glycosylated, glycosylphosphatidylinositol (GPI)-linked protein, is expressed preferentially by myofiber synaptic nuclei in embryonic and adult muscle, and that CD24 expression is restricted to the central region of muscle independent of innervation. Moreover, we show that CD24 has a role in presynaptic differentiation, because synaptic transmission is depressed and fails entirely, in a cyclical manner, after repetitive stimulation of motor axons in CD24 mutant mice. These deficits in synaptic transmission, which are accompanied by aberrant stimulus-dependent uptake of AM1-43 from axons, indicate that CD24 is required for normal presynaptic maturation and function. Because CD24 is also expressed in some neurons, additional experiments will be required to determine whether pre- or postsynaptic CD24 mediates these effects on presynaptic development and function.

Animals↗

PubMed2006

Expression of MuSK in in vitro-innervated human muscle.

Unlike rodent or avian muscle, which forms clusters of acetylcholine receptors (AChRs) on its surface, exhibits cross striations, and contracts spontaneously even if cultured in the absence of the nerve, human muscle must be innervated to reach such differentiation level under in vitro conditions (Kobayashi and Askanas, 1985; Mars et al., 2001). Because it is known that AChR clustering and other aspects of neuromuscular junction (NMJ) formation necessitate the activation of muscle-specific kinase (MuSK), one explanation of this inability of human muscle is that it has no MuSK or that it cannot be activated in the absence of the nerve. To test this hypothesis we analyzed cultured human muscle for the expression of MuSK at two stages of differentiation: postfusion myotube and innervated, contracting myotube. Analyses were carried out at the mRNA level, as no reliable anti-MuSK antibodies are available for the immunocytochemical demonstration of MuSK in cultured human muscle. The presence of MuSK, however, can be tested indirectly, as it can be activated in the absence of the nerve simply by growing muscle culture on laminin coating (Kummer et al., 2004). In the second part of our study, we therefore tested human myotubes for the presence and activation of MuSK by exposing them to laminin coating and by analyzing them afterwards for the areas of postsynaptic differentiation typical for NMJ formation.

DNA Primers↗

PubMed2006

Microarray screen for synaptic genes in the neuromuscular junction.

The formation of neuromuscular synapses requires a complex exchange of signals between motor neurons and skeletal muscle fibers. Essential for the formation of neuromuscular junction (NMJ) is the activation of MuSK, a muscle-specific receptor tyrosine kinase (DeChiara et al., 1996). In mice lacking MuSK, motor axons fail to stop and differentiate, acetylcholine receptors (AChRs) fail to cluster, and AChR genes are expressed uniformly in muscle (DeChiara et al., 1996; Gautam et al., 1996). The retrograde signals for presynaptic differentiation are not known. Because synapse-specific transcription, like presynaptic differentiation, is MuSK-dependent, it is possible that retrograde signals for presynaptic differentiation might be encoded by genes that are expressed preferentially by synaptic nuclei. To identify such synapse-specific genes we screened Affymetrix microarrays with RNA from the dissected, synapse-enriched, and extrasynaptic regions of skeletal muscle and further studied those genes that encode for the secreted or cell-surface proteins.

Acetylcholinesterase↗

PubMed2006

Effects of acetylcholinesterase gene silencing on its activity in cultured human skeletal muscle.

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.

Acetylcholinesterase↗

PubMed2005

Expression and distribution of acetylcholinesterase among the cellular components of the neuromuscular junction formed in human myotube in vitro.

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.

Acetylcholinesterase↗

PubMed2005

Insulin-induced exocytosis in single, in vitro innervated human muscle fibres: a new approach.

We describe a new approach for studying insulin-induced exocytosis in individual, well-differentiated, innervated human muscle fibres. We used an in vitro system in which motor axons extending from embryonic rat spinal cord explants functionally innervate co-cultured human muscle fibres. Under such conditions, the human muscle fibres reach a high degree of differentiation that is never observed in non-innervated, cultured human muscle fibres. To monitor insulin-induced membrane dynamics, we used confocal microscopy to measure the fluorescence intensity changes of the styryl dye FM1-43, a marker for membrane area. The fluorescence intensity increased after insulin stimulation. This increase, as well as the intensity of staining for the glucose transporter 4 (GLUT4), was significantly higher in the innervated and contracting fibres than in myoblasts and myotubes. This shows that in vitro innervation of human muscle cells not only enhances the differentiation stage but also improves the insulin response. Our approach allows continuous monitoring and quantitative assessment of insulin-induced increase in cumulative exocytosis in individual human muscle fibres at a differentiation level practically corresponding to that of adult muscle. It is therefore a suitable system for studying various parameters affecting the mechanisms underlying insulin-induced GLUT4 translocation in human skeletal muscle.

Animals↗

PubMed2004

Origin of acetylcholinesterase in the neuromuscular junction formed in the in vitro innervated human muscle.

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.

Acetylcholinesterase↗

PubMed2003

Localization of mRNAs encoding acetylcholinesterase and butyrylcholinesterase in the rat spinal cord by nonradioactive in situ hybridization.

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.

Acetylcholinesterase↗