Lindsay M. Angus

Expression of utrophin A mRNA correlates with the oxidative capacity of skeletal muscle fiber types and is regulated by calcineurin/NFAT signaling.

Utrophin levels have recently been shown to be more abundant in slow vs. fast muscles, but the nature of the molecular events underlying this difference remains to be fully elucidated. Here, we determined whether this difference is due to the expression of utrophin A or B, and examined whether transcriptional regulatory mechanisms are also involved. Immunofluorescence experiments revealed that slower fibers contain significantly more utrophin A in extrasynaptic regions as compared with fast fibers. Single-fiber RT-PCR analysis demonstrated that expression of utrophin A transcripts correlates with the oxidative capacity of muscle fibers, with cells expressing myosin heavy chain I and IIa demonstrating the highest levels. Functional muscle overload, which stimulates expression of a slower, more oxidative phenotype, induced a significant increase in utrophin A mRNA levels. Because calcineurin has been implicated in controlling this slower, high oxidative myofiber program, we examined expression of utrophin A transcripts in muscles having altered calcineurin activity. Calcineurin inhibition resulted in an 80% decrease in utrophin A mRNA levels. Conversely, muscles from transgenic mice expressing an active form of calcineurin displayed higher levels of utrophin A transcripts. Electrophoretic mobility shift and supershift assays revealed the presence of a nuclear factor of activated T cells (NFAT) binding site in the utrophin A promoter. Transfection and direct gene transfer studies showed that active forms of calcineurin or nuclear NFATc1 transactivate the utrophin A promoter. Together, these results indicate that expression of utrophin A is related to the oxidative capacity of muscle fibers, and implicate calcineurin and its effector NFAT in this mechanism.

Expression of mutant Ets protein at the neuromuscular synapse causes alterations in morphology and gene expression

The localized transcription of several muscle genes at the motor endplate is controlled by the Ets transcription factor GABP. To evaluate directly its contribution to the formation of the neuromuscular junction, we generated transgenic mice expressing a general Ets dominant‐negative mutant specifically in skeletal muscle. Quantitative RT–PCR analysis demonstrated that the expression of genes containing an Ets‐binding site was severely affected in the mutant mice. Conversely, the expression of other synaptic genes, including MuSK and Rapsyn, was unchanged. In these animals, muscles expressing the mutant transcription factor developed normally, but examination of the post‐synaptic morphology revealed marked alterations of both the primary gutters and secondary folds of the neuromuscular junction. Our results demonstrate that Ets transcription factors are crucial for the normal formation of the neuromuscular junction. They further show that Ets‐independent mechanisms control the synaptic expression of a distinct set of synaptic genes.

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.