Identifying FDA-Approved Drugs that Upregulate Utrophin A as a Therapeutic Strategy for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations and deletions within the DMD gene, which result in a lack of dystrophin protein at the sarcolemma of skeletal muscle fibers. The absence of dystrophin fragilizes the sarcolemma and compromises its integrity during cycles of muscle contraction, which, progressively, leads to reductions in muscle mass and function. DMD is thus a progressive muscle-wasting disease that results in a loss of ambulation, cardiomyopathy , respiratory impairment, and death. Although there is presently no cure for DMD, recent advances have led to many promising treatments. One such approach entails increasing expression of a homologous protein to dystrophin, named utrophin A, which is endogenously expressed in both healthy and DMD muscle fibers. Upregulation of utrophin A all along the sarcolemma of DMD muscle fibers can, in part, compensate for the absence of dystrophin. Over the years, our laboratory has focused a significant portion of our efforts in identifying and characterizing drugs and small molecules for their ability to target utrophin A and cause its overexpression. As part of these efforts, we have recently developed a novel ELISA-based high-throughput drug screen, to identify FDA-approved drugs that increase the expression of utrophin A in muscle cells in culture as well as in dystrophic mice. Here, we describe our overall strategy to identify and characterize several FDA-approved drugs that upregulate utrophin A expression and provide details on all experimental approaches. Such strategy has the potential to lead to the rapid development of novel therapeutics for DMD.

Aberrant NLRP3 Inflammasome Activation Ignites the Fire of Inflammation in Neuromuscular Diseases.

Inflammasomes are molecular hubs that are assembled and activated by a host in response to various microbial and non-microbial stimuli and play a pivotal role in maintaining tissue homeostasis. The NLRP3 is a highly promiscuous inflammasome that is activated by a wide variety of sterile triggers, including misfolded protein aggregates, and drives chronic inflammation via caspase-1-mediated proteolytic cleavage and secretion of proinflammatory cytokines, interleukin-1ß and interleukin-18. These cytokines further amplify inflammatory responses by activating various signaling cascades, leading to the recruitment of immune cells and overproduction of proinflammatory cytokines and chemokines, resulting in a vicious cycle of chronic inflammation and tissue damage. Neuromuscular diseases are a heterogeneous group of muscle disorders that involve injury or dysfunction of peripheral nerves, neuromuscular junctions and muscles. A growing body of evidence suggests that dysregulation, impairment or aberrant NLRP3 inflammasome signaling leads to the initiation and exacerbation of pathological processes associated with neuromuscular diseases. In this review, we summarize the available knowledge about the NLRP3 inflammasome in neuromuscular diseases that affect the peripheral nervous system and amyotrophic lateral sclerosis, which affects the central nervous system. In addition, we also examine whether therapeutic targeting of the NLRP3 inflammasome components is a viable approach to alleviating the detrimental phenotype of neuromuscular diseases and improving clinical outcomes.

Targeting IRES-dependent translation as a novel approach for treating Duchenne muscular dystrophy.

Internal-ribosomal entry sites (IRES) are translational elements that allow the initiation machinery to start protein synthesis via internal initiation. IRESs promote tissue-specific translation in stress conditions when conventional cap-dependent translation is inhibited. Since many IRES-containing mRNAs are relevant to diseases, this cellular mechanism is emerging as an attractive therapeutic target for pharmacological and genetic modulations. Indeed, there has been growing interest over the past years in determining the therapeutic potential of IRESs for several disease conditions such as cancer, neurodegeneration and neuromuscular diseases including Duchenne muscular dystrophy (DMD). IRESs relevant for DMD have been identified in several transcripts whose protein product results in functional improvements in dystrophic muscles. Together, these converging lines of evidence indicate that activation of IRES-mediated translation of relevant transcripts in DMD muscle represents a novel and appropriate therapeutic strategy for DMD that warrants further investigation, particularly to identify agents that can modulate their activity.

Overexpression of Staufen1 in DM1 mouse skeletal muscle exacerbates dystrophic and atrophic features.

In myotonic dystrophy type 1 (DM1), the CUG expansion (CUGexp) in the 3' untranslated region of the dystrophia myotonica protein kinase messenger ribonucleic acid affects the homeostasis of ribonucleic acid-binding proteins, causing the multiple symptoms of DM1. We have previously reported that Staufen1 is increased in skeletal muscles from DM1 mice and patients and that sustained Staufen1 expression in mature mouse muscle causes a progressive myopathy. Here, we hypothesized that the elevated levels of Staufen1 contributes to the myopathic features of the disease. Interestingly, the classic DM1 mouse model human skeletal actin long repeat (HSALR) lacks overt atrophy while expressing CUGexp transcripts and elevated levels of endogenous Staufen1, suggesting a lower sensitivity to atrophic signaling in this model. We report that further overexpression of Staufen1 in the DM1 mouse model HSALR causes a myopathy via inhibition of protein kinase B signaling through an increase in phosphatase tensin homolog, leading to the expression of atrogenes. Interestingly, we also show that Staufen1 regulates the expression of muscleblind-like splicing regulator 1 and CUG-binding protein elav-like family member 1 in wild-type and DM1 skeletal muscle. Together, data obtained from these new DM1 mouse models provide evidence for the role of Staufen1 as an atrophy-associated gene that impacts progressive muscle wasting in DM1. Accordingly, our findings highlight the potential of Staufen1 as a therapeutic target and biomarker.

Identification of therapeutics that target eEF1A2 and upregulate utrophin A translation in dystrophic muscles.

Up-regulation of utrophin in muscles represents a promising therapeutic strategy for the treatment of Duchenne Muscular Dystrophy. We previously demonstrated that eEF1A2 associates with the 5'UTR of utrophin A to promote IRES-dependent translation. Here, we examine whether eEF1A2 directly regulates utrophin A expression and identify via an ELISA-based high-throughput screen, FDA-approved drugs that upregulate both eEF1A2 and utrophin A. Our results show that transient overexpression of eEF1A2 in mouse muscles causes an increase in IRES-mediated translation of utrophin A. Through the assessment of our screen, we reveal 7 classes of FDA-approved drugs that increase eEF1A2 and utrophin A protein levels. Treatment of mdx mice with the 2 top leads results in multiple improvements of the dystrophic phenotype. Here, we report that IRES-mediated translation of utrophin A via eEF1A2 is a critical mechanism of regulating utrophin A expression and reveal the potential of repurposed drugs for treating DMD via this pathway.

Celecoxib treatment improves muscle function in mdx mice and increases utrophin A expression.

Duchenne muscular dystrophy (DMD) is a genetic and progressive neuromuscular disorder caused by mutations and deletions in the dystrophin gene. Although there is currently no cure, one promising treatment for DMD is aimed at increasing endogenous levels of utrophin A to compensate functionally for the lack of dystrophin. Recent studies from our laboratory revealed that heparin treatment of mdx mice activates p38 MAPK, leading to an upregulation of utrophin A expression and improvements in the dystrophic phenotype. Based on these findings, we sought to determine the effects of other potent p38 activators, including the cyclooxygenase (COX)-2 inhibitor celecoxib. In this study, we treated 6-wk-old mdx mice for 4 wk with celecoxib. Immunofluorescence analysis of celecoxib-treated mdx muscles revealed a fiber type switch from a fast to a slower phenotype along with beneficial effects on muscle fiber integrity. In agreement, celecoxib-treated mdx mice showed improved muscle strength. Celecoxib treatment also induced increases in utrophin A expression ranging from ∼1.5- to 2-fold in tibialis anterior diaphragm and heart muscles. Overall, these results highlight that activation of p38 in muscles can indeed lead to an attenuation of the dystrophic phenotype and reveal the potential role of celecoxib as a novel therapeutic agent for the treatment of DMD.-Péladeau, C., Adam, N. J., Jasmin, B. J. Celecoxib treatment improves muscle function in mdx mice and increases utrophin A expression.

A specific FMNL2 isoform is up-regulated in invasive cells.

BACKGROUND: Formins are a highly conserved family of cytoskeletal remodeling proteins. A growing body of evidence suggests that formins play key roles in the progression and spread of a variety of cancers. There are 15 human formin proteins and of these the Diaphanous-Related Formins (DRFs) are the best characterized. Included in the DRFs are the Formin-Like proteins, FMNL1, 2 & 3, each of which have been strongly implicated in driving tumorigenesis and metastasis of specific tumors. In particular, increased FMNL2 expression correlates with increased invasiveness of colorectal cancer (CRC) in vivo and for a variety of CRC cell-lines in vitro. FMNL2 expression is also required for invasive cell motility in other cancer cell-lines. There are multiple alternatively spliced isoforms of FMNL2 and it is predicted that the encoded proteins will differ in their regulation, subcellular localization and in their ability to regulate cytoskeletal dynamics. RESULTS: Using RT-PCR we identified four FMNL2 isoforms expressed in CRC and melanoma cell-lines. We find that a previously uncharacterized FMNL2 isoform is predominantly expressed in a variety of melanoma and CRC cell lines; this isoform is also more effective in driving 3D motility. Building on previous reports, we also show that FMNL2 is required for invasion in A375 and WM266.4 melanoma cells. CONCLUSIONS: Taken together, these results suggest that FMNL2 is likely to be generally required in melanoma cells for invasion, that a specific isoform of FMNL2 is up-regulated in invasive CRC and melanoma cells and this isoform is the most effective at facilitating invasion.

Combinatorial therapeutic activation with heparin and AICAR stimulates additive effects on utrophin A expression in dystrophic muscles.

Upregulation of utrophin A is an attractive therapeutic strategy for treating Duchenne muscular dystrophy (DMD). Over the years, several studies revealed that utrophin A is regulated by multiple transcriptional and post-transcriptional mechanisms, and that pharmacological modulation of these pathways stimulates utrophin A expression in dystrophic muscle. In particular, we recently showed that activation of p38 signaling causes an increase in the levels of utrophin A mRNAs and protein by decreasing the functional availability of the destabilizing RNA-binding protein called K-homology splicing regulatory protein, thereby resulting in increases in the stability of existing mRNAs. Here, we treated 6-week-old mdx mice for 4 weeks with the clinically used anticoagulant drug heparin known to activate p38 mitogen-activated protein kinase, and determined the impact of this pharmacological intervention on the dystrophic phenotype. Our results show that heparin treatment of mdx mice caused a significant ∼1.5- to 3-fold increase in utrophin A expression in diaphragm, extensor digitorum longus and tibialis anterior (TA) muscles. In agreement with these findings, heparin-treated diaphragm and TA muscle fibers showed an accumulation of utrophin A and ß-dystroglycan along their sarcolemma and displayed improved morphology and structural integrity. Moreover, combinatorial drug treatment using both heparin and 5-amino-4-imidazolecarboxamide riboside (AICAR), the latter targeting 5' adenosine monophosphate-activated protein kinase and the transcriptional activation of utrophin A, caused an additive effect on utrophin A expression in dystrophic muscle. These findings establish that heparin is a relevant therapeutic agent for treating DMD, and illustrate that combinatorial treatment of heparin with AICAR may serve as an effective strategy to further increase utrophin A expression in dystrophic muscle via activation of distinct signaling pathways.