Céline Boudreau-Larivière

Molecular Mechanisms Underlying the Activity‐Linked Alterations in Acetylcholinesterase mRNAs in Developing Versus Adult Rat Skeletal Muscles

Abstract: The molecular mechanisms underlying the activity‐linked plasticity of acetylcholinesterase (AChE) mRNA levels in mammalian skeletal muscle have yet to be established. Here, we demonstrate that denervation of adult muscle induces a dramatic (up to 90%) and rapid (within 24 h) decrease in the abundance of AChE mRNAs. By contrast, denervation of 14‐day‐old rats leads to a significantly less pronounced reduction (50% of control) in the expression of AChE mRNAs. Assessment of the transcriptional activity of the AChE gene reveals that it remains essentially unchanged in adult denervated muscles, whereas it displays an approximately two‐ to three‐fold increase (p < 0.05) in denervated muscles from 2‐ to 14‐day‐old rats. In addition, we observed a higher rate of degradation of in vitro transcribed AChE mRNAs upon incubation with protein extracts from denervated muscles. Finally, UV‐crosslinking experiments reveal that denervation increases the abundance of RNA‐protein interactions in the 3′ untranslated region of AChE transcripts. Taken together, these data suggest that the abundance of AChE transcripts in mature muscles is controlled primarily via posttranscriptional regulatory mechanisms, whereas in neo‐ and postnatal muscles, both transcriptional and posttranscriptional regulation appears critical in dictating AChE mRNA levels. Accordingly, the activity‐linked transcriptional regulation of the AChE gene appears to demonstrate a high level of plasticity during muscle development when maturation of the neuromuscular junctions is still occurring.

CALCITONIN GENE-RELATED PEPTIDE DECREASES EXPRESSION OF ACETYLCHOLINESTERASE IN MAMMALIAN MYOTUBES

Nerve‐derived trophic factors are known to modulate expression of acetylcholinesterase (AChE) in skeletal muscle fibers, yet the precise identity of these factors remains elusive. In the present study, we treated mouse C2 myotubes with calcitonin gene‐related peptide (CGRP). Compared to non‐treated myotubes, cell‐associated AChE activity levels were decreased by ∼60% after 48 h of treatment. A parallel reduction in AChE total protein levels was also observed as determined by Western blot analysis. The reduction in AChE activity was due to a decrease in the levels of the G1 molecular form and to an elimination of G4. By contrast, levels of secreted AChE remained unchanged following CGRP treatment. Finally, the overall decrease in AChE activity was accompanied by a reduction in AChE transcripts which could not be attributed to changes in the transcriptional rate of the ACHE gene.

Ciliary neurotrophic factor: regulation of acetylcholinesterase in skeletal muscle and distribution of messenger RNA encoding its receptor in synaptic versus extrasynaptic compartments

Several recent studies have shown that the ciliary neurotrophic factor exerts myotrophic effects in addition to its well-characterized neurotrophic actions on various neuronal populations. Since expression of acetylcholinesterase in skeletal muscle has been shown to be regulated by putative yet unknown nerve-derived trophic factors, we tested the hypothesis that the ciliary neurotrophic factor is a neurotrophic agent capable of influencing expression of acetylcholinesterase in adult rat skeletal muscle in vivo. To this end, we first determined the impact of daily ciliary neurotrophic factor administration on expression of acetylcholinesterase in both intact and denervated rat soleus muscles. The results of our experiments indicate that although chronic administration of ciliary neurotrophic factor partially counteracted the atrophic response of soleus muscles to surgical denervation, thus confirming its myotrophic effects, it failed to either increase acetylcholinesterase expression in intact muscles or prevent the decrease normally occurring in seven-day denervated muscles. In fact, acetylcholinesterase messenger RNA and enzyme levels were further reduced by ciliary neurotrophic factor treatment in denervated muscles without significant modifications in the pattern of acetylcholinesterase molecular forms. Conversely, transcript levels of the epsilon subunit of the acetylcholine receptor in intact and denervated soleus muscles treated with the ciliary neurotrophic factor were similar to those observed in their respective counterparts from vehicle-treated animals. In addition, we also determined whether transcripts encoding the receptor for the ciliary neurotrophic factor selectively accumulate in junctional domains of rat skeletal muscle fibres. In contrast to the preferential localization of transcripts encoding acetylcholinesterase and the epsilon subunit of the acetylcholine receptor within the postsynaptic sarcoplasm, messenger RNAs for the ciliary neurotrophic factor receptor appeared homogeneously distributed between junctional and extra-junctional compartments of both diaphragm and extensor digitorum longus muscle fibres, with no compelling evidence for a selective accumulation within the postsynaptic sarcoplasm. These data show that the ciliary neurotrophic factor exerts an inhibitory influence on expression of acetylcholinesterase in muscle fibres. Furthermore, the lack of an effect on expression of the epsilon acetylcholine receptor transcripts indicates that treatment with ciliary neurotrophic factor does not lead to general adaptations in the expression of all synaptic proteins. Given the distribution of transcripts encoding the ciliary neurotrophic factor receptor along multinucleated muscle fibres, we propose a model whereby the ciliary neurotrophic factor, or a related unknown molecule that also utilizes the receptor for the ciliary neurotrophic factor, contributes to the maintenance of low levels of enzyme activity in extrajunctional regions of muscle fibres by acting as a repressor of acetylcholinesterase expression that functions directly or indirectly via a pretranslational regulatory mechanism. Accordingly, these results further highlight the complexity of the regulatory mechanisms presiding over acetylcholinesterase expression in vivo.

Molecular Mechanisms Controlling the Synapse-Specific Expression and Activity-Linked Regulation of Acetylcholinesterase in Skeletal Muscle Fibers

Acetylcholinesterase (AChE) is mostly known for its pivotal role in the inactivation of acetylcholine at cholinergic synapses in both central and peripheral nervous systems. This enzyme displays a rich polymorphism since it exists as a variety of molecular forms that may be classified as either homomeric or heteromeric on the basis of their association with specialized structural subunits. Homomeric forms include the G, monomer and G2 di-mer as well as a glycophospholipid-linked (GPI) dimer. Conversely, heteromers consist of: i) the asymmetric forms A4, A8 or A12 in which 1, 2 or 3 soluble G4 tetramers attach to a collagenic structural subunit, respectively; and ii) amphiphilic tetramers G4 linked to a 20 kDa hydrophobic anchor. Although the functional significance of this polymorphism is still elusive, it has been suggested that it allows the placement of catalytically active subunits in distinct cell types and subcellular locations where each form can assume site-specific functions. In mammals for example, the asymmetric forms of AChE are exclusively expressed in differentiated muscle and neuronal cells whereas GPI-linked dimers are found preferentially in tissues of hematopoietic origin. Such varied patterns of expression suggest that expression of AChE involves several levels of regulatory mechanisms ranging from tissue-specific transcriptional control to highly regulated post-translational events.

Expression of the Acetylcholinesterase Gene in Skeletal Muscle Fibers

In contrast to extrasynaptic compartments of skeletal muscle fibers, mRNAs encoding acetylcholinesterase (AChE) are ~ 10-fold more abundant in the postsynaptic sarcoplasm (1,2) where their expression is markedly influenced by nerve-evoked electrical activity (2,3). In order to understand the molecular events involved in the regulation of AChE expression during synapse formation in embryonic and neo-natal muscles, we have cloned a 4.7 kb DNA fragment upstream of the translation start site in the rat AChE gene and generated multiple promoter-reporter gene constructs containing LacZ and a nuclear localization signal (nlsLacZ). These constructs were directly injected into tibialis anterior muscles of mice and 14 days later, muscles were excised and frozen. Muscles were subsequently cut in a cryostat and tissue sections were histochemically stained for the demonstration of β-galactosidase. The position of blue myonuclei indicative of promoter activity, was compared to that of neuromuscular junctions identified by AChE histochemistry. Injections of promoter-reporter gene constructs containing DNA fragments ranging from 4.7 to 1.5 kb led to a strong level of expression within muscle fibers. Surprisingly, quantitative analysis further revealed that expression of nlsLacZ was not confined to synaptic areas despite the presence of an N-box motif in the promoter region shown recently to play a crucial role in directing synapse-specific expression of other genes encoding synaptic proteins (4,5). Deletion of 600 bp in intron 1 from the 1.5 kb DNA fragment completely abolished muscle expression. In addition, transfection of motoneurons with these two latter constructs showed that both were equally effective in driving expression of LacZ thereby indicating the presence of cis-acting regulatory elements essential for muscle expression in the first intron of the AChE gene.

Activity-Linked Regulation of Acetylcholinesterase mRNA Levels Involves Distinct Molecular Mechanisms in Developing Versus Adult Skeletal Muscles

Despite the wealth of information available on the plasticity of acetylcholinesterase (AChE) molecular forms subjected to altered levels of neuromuscular activation, our knowledge of the cellular and molecular mechanisms involved in the localization and activity-linked regulation of AChE in skeletal muscle is still rudimentary. Levels of AChE mRNA for instance, are known to be highly sensitive to nerve-evoked electrical activity (1,2) but the underlying molecular events have yet to be clearly determined. In the present study, we therefore examined the contribution of transcriptional and post-transcriptional regulatory mechanisms in the control of AChE mRNA levels in denervated neo-natal and adult rat skeletal muscles. Our results show that in comparison to the sharp increase (20-fold) in the levels of transcripts encoding the α-subunit of the acetylcholine receptor (AChR), denervation of adult muscle induced a rapid (within 2 days) and large (8-fold) decrease in the abundance of AChE mRNAs. Northern blot analysis also revealed that the two predominant species of AChE T transcripts expressed in adult muscle are reduced to a similar extent by muscle denervation. Furthermore, nuclear run-on assays showed that the transcriptional activity of the AChE gene and, unexpectedly, of the AChR α-subunit gene remained essentially unchanged following denervation indicating that post-transcriptional events are responsible for the observed modifications in AChE and AChR α-subunit transcript levels. In separate experiments, we examined whether denervation resulted in significant changes in the half-life of these transcripts by injecting animals with actinomycin D. Quantitative analysis revealed that Egr-1 transcripts, used as a positive control in these experiments, was turning over rapidly (~ 2.5 hr) in skeletal muscle.

Neuromuscular Factors Influencing Acetylcholinesterase Gene Expression in Skeletal Muscle Fibers

Acetylcholinesterase (AChE) is of particular interest with regards to muscle plasticity since levels of AChE molecular forms are known to be highly sensitive to neural influences. For example, muscle paralysis induced via surgical denervation results in general in the rapid disappearance of the synaptic collagen-like tailed AChE forms (Massoulie et al., 1993). Alternatively, enhanced neuromuscular activation achieved by exercise training programs and compensatory hypertrophy lead to significant increases in whole muscle AChE activity which are reflected by specific and prominent changes in the levels of the various molecular forms (Fernandez and Donoso, 1988; Jasmin and Gisiger, 1990; Gisiger et al., 1991; Jasmin et al., 1991; Sveistrup et al., 1994). Despite the wealth of information available on the plasticity of AChE molecular forms confronted with altered levels of neuromuscular activation, our knowledge of the cellular and molecular basis underlying the activity-linked regulation of AChE in muscle is still rudimentary. In this context, several levels of regulatory mechanisms including transcriptional, post-transcriptional as well as post-translational may be envisaged. Within the last few years, several laboratories have succeeded in isolating cDNA and genomic clones encoding AChE in a variety of species (Schumacher et al., 1986; Sikorav et al., 1987; Rotundo et al., 1988; Maulet et al., 1990; Rachinsky et al., 1990; Li et al., 1991; Legay et al., 1993a) thus allowing for the study of the cellular and molecular mechanisms involved in the regulation and localization of AChE at the nucleic acid level.