A 1.3 kb promoter fragment confers spatial and temporal expression of utrophin A mRNA in mouse skeletal muscle fibers.

Upregulation of utrophin in muscle is currently being examined as a potential therapy for Duchenne muscular dystrophy patients. In this context, we generated transgenic mice harboring a 1.3 kb human utrophin A promoter fragment driving expression of the lacZ gene. Characterization of reporter expression during postnatal muscle development revealed that the levels and localization of beta-galactosidase parallel expression of utrophin A transcripts. Moreover, we noted that the utrophin A promoter is more active in slow soleus muscles. Additionally, expression of the reporter gene was regulated during muscle regeneration in a manner similar to utrophin A transcripts. Together, these results show that the utrophin A promoter-lacZ construct mirrors expression of utrophin A mRNAs indicating that this utrophin A promoter fragment confers temporal and spatial patterns of expression in skeletal muscle. This transgenic mouse will be valuable as an in vivo model for developing and testing molecules aimed at increasing utrophin A expression.

Accumulation of Nav1 mRNAs at differentiating postsynaptic sites in rat soleus muscles.

Acetylcholine receptors (AChRs) and voltage-gated sodium channels (Na(V)1s) accumulate at different times in the development of the murine neuromuscular junction (NMJ). We used in situ hybridization to study the relationship of Na(V)1 mRNA accumulation to this difference. mRNAs encoding both muscle Na(V)1 isoforms, Na(v)1.4 and Na(v)1.5, were first concentrated at NMJs at birth, when the proteins start to accumulate. Within 4 weeks, Na(v)1.4 mRNA increased 5-fold at the NMJ while Na(v)1.5 mRNA became undetectable. Na(V)1 mRNA accumulation occurred even if the nerve was cut at birth. Like AChR mRNA, Na(V)1 mRNA accumulated at denervated synaptic sites on regenerating muscles and in response to ectopically expressed neural agrin. Clustering of Na(V)1 at the NMJ follows that of its mRNA while AChR clustering precedes its mRNA clustering by several days. This suggests that factors other than local mRNA upregulation determine the timing of clustering of these two important postsynaptic ion channels.

Localization of the RNA-binding proteins Staufen1 and Staufen2 at the mammalian neuromuscular junction.

Staufen is an RNA-binding protein, first identified for its role in oogenesis and CNS development in Drosophila. Two mammalian homologs of Staufen have been identified and shown to bind double-stranded RNA and tubulin, and to function in the somatodendritic transport of mRNA in neurons. Here, we examined whether Staufen proteins are expressed in skeletal muscle in relation to the neuromuscular junction. Immunofluorescence experiments revealed that Staufen1 (Stau1) and Staufen2 (Stau2) accumulate preferentially within the postsynaptic sarcoplasm of muscle fibers as well as at newly formed ectopic synapses. Western blot analyses showed that the levels of Stau1 and Stau2 are greater in slow muscles than in fast-twitch muscles. Muscle denervation induced a significant increase in the expression of Stau1 and Stau2 in the extrasynaptic compartment of both fast and slow muscles. Consistent with these observations, we also demonstrated that expression of Stau1 and Stau2 is increased during myogenic differentiation and that treatment of myotubes with agrin and neuregulin induces a further increase in the expression of both Staufen proteins. We propose that Stau1 and Stau2 are key components of the postsynaptic apparatus in muscle, and that they contribute to the maturation and plasticity of the neuromuscular junction.

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.

Voltage-gated sodium channels and ankyrinG occupy a different postsynaptic domain from acetylcholine receptors from an early stage of neuromuscular junction maturation in rats.

Spatial segregation of membrane proteins is a feature of many excitable cells. In skeletal muscle, clusters of acetylcholine receptors (AChRs) and voltage-gated sodium channels (Na(V)1s) occupy distinct domains at the neuromuscular junction (NMJ). We used quantitative immunolabeling of developing rat soleus muscles to study the mechanism of ion channel segregation and Na(V)1 clustering at NMJs. When Na(V)1s can first be detected, at birth, they already occupy a postsynaptic domain that is distinct from that occupied by AChRs. At this time, Na(V)1s are expressed only in a diffuse area that extends 50-100 microm from the immature NMJ. However, in the region of the high-density AChR cluster at NMJ itself, Na(V)1s are actually present in lower density than in the immediately surrounding membrane. These distinctive features of the Na(V)1 distribution at birth are closely correlated with the distribution of ankyrinG immunolabeling. This suggests that an interaction with ankyrinG plays a role in the initial segregation of Na(V)1s from AChRs. Both Na(V)1 and ankyrinG become clustered at the NMJ itself 1-2 weeks after birth, coincident with the formation of postsynaptic folds. Syntrophin immunolabeling codistributes with AChRs and never resembles that for Na(V)1 or ankyrinG. Therefore, syntrophin is unlikely to play an important part in the initial accumulation of Na(V)1 at the NMJ. These findings suggest that the segregation of Na(V)1 from AChRs begins early in NMJ formation and occurs as a result of the physical exclusion of Na(V)1 and ankyrinG from the region of nerve-muscle contact rather than by a process of active clustering.

Multiple regulatory events controlling the expression and localization of utrophin in skeletal muscle fibers: insights into a therapeutic strategy for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most prevalent inherited muscle disease and results from mutations/deletions in the X-linked dystrophin gene. Although several approaches have been envisaged to counteract the effects of this progressive disease, there is currently no cure available. One strategy consists in utilizing a protein normally expressed in DMD muscle which, once expressed at appropriate levels and at the correct subcellular location, could compensate for the lack of dystrophin. A candidate for such a role is the dystrophin-related protein now referred to as utrophin. In contrast to dystrophin, which is expressed along the length of healthy muscle fibers, utrophin accumulates at the neuromuscular junction in both normal and DMD fibers. Several years ago, we began a series of experiments to determine the mechanisms responsible for the expression of utrophin at the neuromuscular synapse. Initially, we showed that utrophin transcripts accumulate preferentially within the postsynaptic sarcoplasm. To determine whether selective transcription of the utrophin gene accounts for this synaptic accumulation of utrophin mRNAs, we injected several utrophin promoter-reporter constructs directly into mouse muscle and demonstrated the preferential synaptic expression of the reporter gene. These results suggested that local transcriptional activation of the utrophin gene is responsible for the accumulation of utrophin mRNAs at the neuromuscular junction. In these studies, we also demonstrated that an N-box motif contained within the utrophin promoter plays a critical role in directing the synapse-specific expression of the utrophin gene. Additionally, our studies have shown that the ets-factors GABP alpha and beta are part of a protein complex that can bind to the N-box motif to transactivate the gene in muscle cells in culture and in vivo. In these experiments, we also noted that the nerve-derived trophic factors agrin and ARIA/heregulin regulate expression of utrophin via the activation of GABP alpha and beta which in turn, transactivate the utrophin gene via the N-box motif. Although these studies demonstrate that transcriptional activation can regulate utrophin mRNA levels, it is possible that additional mechanisms are also involved. In particular, the association of mRNAs with cytoskeletal elements and RNA-binding proteins may contribute to the accumulation of utrophin transcripts within the postsynaptic sarcoplasm. In recent studies, we have begun to examine this and we have now identified specific regions within the 3' untranslated region that are necessary for targeting and stabilizing utrophin mRNAs in skeletal muscle cells. A series of in vivo studies have also led us to conclude that post-transcriptional mechanisms are indeed important in regulating the abundance of utrophin transcripts in muscle. Together, these studies should lead to the identification of cis- and trans-acting elements regulating transcription of the utrophin gene as well as the stability and targeting of its mRNA in muscle cells. The results should therefore, identify specific targets that may become important in designing specific pharmacological interventions directed at increasing the expression of utrophin into extrasynaptic regions of DMD muscle fibers. In addition, these findings will contribute to our basic understanding of the cellular and molecular events involved in the formation, maintenance and plasticity of the neuromuscular synapse.