Guy Bélanger

Pharmacological activation of PPARβ/δ stimulates utrophin A expression in skeletal muscle fibers and restores sarcolemmal integrity in mature mdx mice

A therapeutic strategy to treat Duchenne muscular dystrophy (DMD) involves identifying compounds that can elevate utrophin A expression in muscle fibers of affected patients. The dystrophin homologue utrophin A can functionally substitute for dystrophin when its levels are enhanced in the mdx mouse model of DMD. Utrophin A expression in skeletal muscle is regulated by mechanisms that promote the slow myofiber program. Since activation of peroxisome proliferator-activated receptor (PPAR) beta/delta promotes the slow oxidative phenotype in skeletal muscle, we initiated studies to determine whether pharmacological activation of PPARbeta/delta provides functional benefits to the mdx mouse. GW501516, a PPARbeta/delta agonist, was found to stimulate utrophin A mRNA levels in C2C12 muscle cells through an element in the utrophin A promoter. Expression of PPARbeta/delta was greater in skeletal muscles of mdx versus wild-type mice. We treated 5-7-week-old mdx mice with GW501516 for 4 weeks. This treatment increased the percentage of muscle fibers expressing slower myosin heavy chain isoforms and stimulated utrophin A mRNA levels leading to its increased expression at the sarcolemma. Expression of alpha1-syntrophin and beta-dystroglycan was restored to the sarcolemma. Improvement of mdx sarcolemmal integrity was evidenced by decreased intracellular IgM staining and decreased in vivo Evans blue dye (EBD) uptake. GW501516 treatment also conferred protection against eccentric contraction (ECC)-induced damage of mdx skeletal muscles, as shown by a decreased contraction-induced force drop and reduction of dye uptake during ECC. These results demonstrate that pharmacological activation of PPARbeta/delta might provide functional benefits to DMD patients through enhancement of utrophin A expression.

Distinct regions in the 3′ untranslated region are responsible for targeting and stabilizing utrophin transcripts in skeletal muscle cells

In this study, we have sought to determine whether utrophin transcripts are targeted to a distinct subcellular compartment in skeletal muscle cells, and have examined the role of the 3′ untranslated region (UTR) in regulating the stability and localization of utrophin transcripts. Our results show that utrophin transcripts associate preferentially with cytoskeleton-bound polysomes via actin microfilaments. Because this association is not evident in myoblasts, our findings also indicate that the localization of utrophin transcripts with cytoskeleton-bound polysomes is under developmental influences. Transfection of LacZ reporter constructs containing the utrophin 3′UTR showed that this region is critical for targeting chimeric mRNAs to cytoskeleton-bound polysomes and controlling transcript stability. Deletion studies resulted in the identification of distinct regions within the 3′UTR responsible for targeting and stabilizing utrophin mRNAs. Together, these results illustrate the contribution of posttranscriptional events in the regulation of utrophin in skeletal muscle. Accordingly, these findings provide novel targets, in addition to transcriptional events, for which pharmacological interventions may be envisaged to ultimately increase the endogenous levels of utrophin in skeletal muscle fibers from Duchenne muscular dystrophy (DMD) patients.

The RNA-binding protein Staufen1 is increased in DM1 skeletal muscle and promotes alternative pre-mRNA splicing

Staufen1 interacts with mRNAs with expanded CUG repeats and promotes their nuclear export and translation, while also promoting alternative splicing of other mRNAs.

Brain-derived neurotrophic factor expression is repressed during myogenic differentiation by miR-206.

J. Neurochem. (2012) 120, 230–238.

Modulation of utrophin A mRNA stability in fast versus slow muscles via an AU-rich element and calcineurin signaling

We examined the role of post-transcriptional mechanisms in controlling utrophin A mRNA expression in slow versus fast skeletal muscles. First, we determined that the half-life of utrophin A mRNA is significantly shorter in the presence of proteins isolated from fast muscles. Direct plasmid injection experiments using reporter constructs containing the full-length or truncated variants of the utrophin 3′UTR into slow soleus and fast extensor digitorum longus muscles revealed that a region of 265 nucleotides is sufficient to confer lower levels of reporter mRNA in fast muscles. Further analysis of this region uncovered a conserved AU-rich element (ARE) that suppresses expression of reporter mRNAs in cultured muscle cells. Moreover, stability of reporter mRNAs fused to the utrophin full-length 3′UTR was lower in the presence of fast muscle protein extracts. This destabilization effect seen in vivo was lost upon deletion of the conserved ARE. Finally, we observed that calcineurin signaling affects utrophin A mRNA stability through the conserved ARE. These results indicate that ARE-mediated mRNA decay is a key mechanism that regulates expression of utrophin A mRNA in slow muscle fibers. This is the first demonstration of ARE-mediated mRNA decay regulating the expression of a gene associated with the slow myogenic program.

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.

The RNA-binding protein Staufen1 impairs myogenic differentiation via a c-myc–dependent mechanism

The expression pattern of Staufen1 during mouse skeletal muscle development is described. Sustained expression of Staufen1 in myoblasts prevents normal differentiation by causing decreases in the expression of key myogenic markers by an SMD-independent mechanism and by promoting the translational regulation of c-myc.

Converging pathways involving microRNA-206 and the RNA-binding protein KSRP control post-transcriptionally utrophin A expression in skeletal muscle

Several reports have previously highlighted the potential role of miR-206 in the post-transcriptional downregulation of utrophin A in cultured cells. Along those lines, we recently identified K-homology splicing regulator protein (KSRP) as an important negative regulator in the post-transcriptional control of utrophin A in skeletal muscle. We sought to determine whether these two pathways act together to downregulate utrophin A expression in skeletal muscle. Surprisingly, we discovered that miR-206 overexpression in cultured cells and dystrophic muscle fibers causes upregulation of endogenous utrophin A levels. We further show that this upregulation of utrophin A results from the binding of miR-206 to conserved sites located in the 3′-UTR (untranslated region) of KSRP, thus causing the subsequent inhibition of KSRP expression. This miR-206-mediated decrease in KSRP levels leads, in turn, to an increase in the expression of utrophin A due to a reduction in the activity of this destabilizing RNA-binding protein. Our work shows that miR-206 can oscillate between direct repression of utrophin A expression via its 3′-UTR and activation of its expression through decreased availability of KSRP and interactions with AU-rich elements located within the 3′-UTR of utrophin A. Our study thus reveals that two apparent negative post-transcriptional pathways can act distinctively as molecular switches causing repression or activation of utrophin A expression.

Staufen1 impairs stress granule formation in skeletal muscle cells from myotonic dystrophy type 1 patients

The formation of stress granules (SGs) in proliferating, quiescent, and differentiated muscle cells is examined. DM1 myoblasts fail to properly form SGs in response to stress, thereby likely contributing to the complex DM1 pathogenesis. Staufen1 participates in the regulation of SG formation in DM1 myoblasts.

HuR Mediates Changes in the Stability of AChR β-Subunit mRNAs after Skeletal Muscle Denervation

Acetylcholine receptors (AChRs) are heteromeric membrane proteins essential for neurotransmission at the neuromuscular junction. Previous work showed that muscle denervation increases expression of AChR mRNAs due to transcriptional activation of AChR subunit genes. However, it remains possible that post-transcriptional mechanisms are also involved in controlling the levels of AChR mRNAs following denervation. We examined whether post-transcriptional events indeed regulate AChR β-subunit mRNAs in response to denervation. First, in vitro stability assays revealed that the half-life of AChR β-subunit mRNAs was increased in the presence of denervated muscle protein extracts. A bioinformatics analysis revealed the existence of a conserved AU-rich element (ARE) in the 3′-untranslated region (UTR) of AChR β-subunit mRNA. Furthermore, denervation of mouse muscle injected with a luciferase reporter construct containing the AChR β-subunit 3′UTR, caused an increase in luciferase activity. By contrast, mutation of this ARE prevented this increase. We also observed that denervation increased expression of the RNA-binding protein human antigen R (HuR) and induced its translocation to the cytoplasm. Importantly, HuR binds to endogenous AChR β-subunit transcripts in cultured myotubes and in vivo, and this binding is increased in denervated versus innervated muscles. Finally, p38 MAPK, a pathway known to activate HuR, was induced following denervation as a result of MKK3/6 activation and a decrease in MKP-1 expression, thereby leading to an increase in the stability of AChR β-subunit transcripts. Together, these results demonstrate the important contribution of post-transcriptional events in regulating AChR β-subunit mRNAs and point toward a central role for HuR in mediating synaptic gene expression. SIGNIFICANCE STATEMENT Muscle denervation is a convenient model to examine expression of genes encoding proteins of the neuromuscular junction, especially acetylcholine receptors (AChRs). Despite the accepted model of AChR regulation, which implicates transcriptional mechanisms, it remains plausible that such events cannot fully account for changes in AChR expression following denervation. We show that denervation increases expression of the RNA-binding protein HuR, which in turn, causes an increase in the stability of AChR β-subunit mRNAs in denervated muscle. Our findings demonstrate for the first time the contribution of post-transcriptional events in controlling AChR expression in skeletal muscle, and points toward a central role for HuR in mediating synaptic development while also paving the way for developing RNA-based therapeutics for neuromuscular diseases.