RESUMO
Targeting AMP-activated protein kinase (AMPK) is emerging as a promising strategy for treating myotonic dystrophy type 1 (DM1), the most prevalent form of adult-onset muscular dystrophy. We previously demonstrated that 5-aminomidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) and exercise, two potent AMPK activators, improve disease features in DM1 mouse skeletal muscles. Here, we employed a combinatorial approach with these AMPK activators and examined their joint impact on disease severity in male and female DM1 mice. Our data reveal that swimming exercise additively enhances the effect of AICAR in mitigating the nuclear accumulation of toxic CUGexp RNA foci. In addition, our findings show a trend towards an enhanced reversal of MBNL1 sequestration and correction in pathogenic alternative splicing events. Our results further demonstrate that the combinatorial impact of exercise and AICAR promotes muscle fiber hypertrophy in DM1 skeletal muscle. Importantly, these improvements occur in a sex-specific manner with greater benefits observed in female DM1 mice. Our findings demonstrate that combining AMPK-activating interventions may prove optimal for rescuing the DM1 muscle phenotype and uncover important sex differences in the response to AMPK-based therapeutic strategies in DM1 mice.
Assuntos
Distrofia Miotônica , Condicionamento Físico Animal , Animais , Feminino , Masculino , Camundongos , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Músculo Esquelético/metabolismo , Distrofia Miotônica/tratamento farmacológico , Distrofia Miotônica/genética , Ribonucleotídeos/farmacologiaRESUMO
Spinal muscular atrophy (SMA) is characterized by the loss of alpha motor neurons in the spinal cord and a progressive muscle weakness and atrophy. SMA is caused by loss-of-function mutations and/or deletions in the survival of motor neuron (SMN) gene. The role of SMN in motor neurons has been extensively studied, but its function and the consequences of its loss in muscle have also emerged as a key aspect of SMA pathology. In this study, we explore the molecular mechanisms involved in muscle defects in SMA. First, we show in C2C12 myoblasts, that arginine methylation by CARM1 controls myogenic differentiation. More specifically, the methylation of HuR on K217 regulates HuR levels and subcellular localization during myogenic differentiation, and the formation of myotubes. Furthermore, we demonstrate that SMN and HuR interact in C2C12 myoblasts. Interestingly, the SMA-causing E134K point mutation within the SMN Tudor domain, and CARM1 depletion, modulate the SMN-HuR interaction. In addition, using the Smn2B/- mouse model, we report that CARM1 levels are markedly increased in SMA muscles and that HuR fails to properly respond to muscle denervation, thereby affecting the regulation of its mRNA targets. Altogether, our results show a novel CARM1-HuR axis in the regulation of muscle differentiation and plasticity as well as in the aberrant regulation of this axis caused by the absence of SMN in SMA muscle. With the recent developments of therapeutics targeting motor neurons, this study further indicates the need for more global therapeutic approaches for SMA.
Assuntos
Atrofia Muscular Espinal , Animais , Modelos Animais de Doenças , Proteína Semelhante a ELAV 1 , Camundongos , Neurônios Motores/metabolismo , Músculos/metabolismo , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genéticaRESUMO
Myotonic dystrophy type 1 (DM1), the most common form of adult muscular dystrophy, is caused by an abnormal expansion of CTG repeats in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The expanded repeats of the DMPK mRNA form hairpin structures in vitro, which cause misregulation and/or sequestration of proteins including the splicing regulator muscleblind-like 1 (MBNL1). In turn, misregulation and sequestration of such proteins result in the aberrant alternative splicing of diverse mRNAs and underlie, at least in part, DM1 pathogenesis. It has been previously shown that disaggregating RNA foci repletes free MBNL1, rescues DM1 spliceopathy, and alleviates associated symptoms such as myotonia. Using an FDA-approved drug library, we have screened for a reduction of CUG foci in patient muscle cells and identified the HDAC inhibitor, vorinostat, as an inhibitor of foci formation; SERCA1 (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) spliceopathy was also improved by vorinostat treatment. Vorinostat treatment in a mouse model of DM1 (human skeletal actin-long repeat; HSALR) improved several spliceopathies, reduced muscle central nucleation, and restored chloride channel levels at the sarcolemma. Our in vitro and in vivo evidence showing amelioration of several DM1 disease markers marks vorinostat as a promising novel DM1 therapy.
Assuntos
Distrofia Miotônica , Splicing de RNA , Vorinostat , Adulto , Animais , Humanos , Camundongos , Processamento Alternativo/efeitos dos fármacos , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Distrofia Miotônica/genética , Splicing de RNA/efeitos dos fármacos , RNA Mensageiro/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Expansão das Repetições de Trinucleotídeos , Vorinostat/metabolismoRESUMO
Myotonic dystrophy type 1 (DM1) is a multisystemic disorder with variable clinical features. Currently, there is no cure or effective treatment for DM1. The disease is caused by an expansion of CUG repeats in the 3' UTR of DMPK mRNAs. Mutant DMPK mRNAs accumulate in nuclei as RNA foci and trigger an imbalance in the level and localization of RNA-binding proteins causing the characteristic missplicing events that account for the varied DM1 symptoms, a disease mechanism referred to as RNA toxicity. In recent years, multiple signalling pathways have been identified as being aberrantly regulated in skeletal muscle in response to the CUG expansion, including AMPK, a sensor of energy status, as well as a master regulator of cellular energy homeostasis. Converging lines of evidence highlight the benefits of activating AMPK signalling pharmacologically on RNA toxicity, as well as on muscle histology and function, in preclinical DM1 models. Importantly, a clinical trial with metformin, an activator of AMPK, resulted in functional benefits in DM1 patients. In addition, exercise, a known AMPK activator, has shown promising effects on RNA toxicity and muscle function in DM1 mice. Finally, clinical trials involving moderate-intensity exercise also induced functional benefits for DM1 patients. Taken together, these studies clearly demonstrate the molecular, histological and functional benefits of AMPK activation and exercise-based interventions on the DM1 phenotype. Despite these advances, several key questions remain; in particular, the extent of the true implication of AMPK in the observed beneficial improvements, as well as how, mechanistically, activation of AMPK signalling improves the DM1 pathophysiology.
Assuntos
Distrofia Miotônica , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Camundongos , Músculo Esquelético/metabolismo , Distrofia Miotônica/tratamento farmacológico , Distrofia Miotônica/genética , RNA Mensageiro/metabolismo , Expansão das Repetições de TrinucleotídeosRESUMO
The development and regeneration of skeletal muscle are mediated by satellite cells (SCs), which ensure the efficient formation of myofibers while repopulating the niche that allows muscle repair following injuries. Pannexin 1 (Panx1) channels are expressed in SCs and their levels increase during differentiation in vitro, as well as during skeletal muscle development and regeneration in vivo. Panx1 has recently been shown to regulate muscle regeneration by promoting bleb-based myoblast migration and fusion. While skeletal muscle is largely influenced in a sex-specific way, the sex-dependent roles of Panx1 in regulating skeletal muscle and SC function remain to be investigated. Here, using global Panx1 knockout (KO) mice, we demonstrate that Panx1 loss reduces muscle fiber size and strength, decreases SC number, and alters early SC differentiation and myoblast fusion in male, but not in female mice. Interestingly, while both male and female Panx1 KO mice display an increase in the number of regenerating fibers following acute injury, the newly formed fibers in male Panx1 KO mice are smaller. Overall, our results demonstrate that Panx1 plays a significant role in regulating muscle development, regeneration, and SC number and function in male mice and reveal distinct sex-dependent functions of Panx1 in skeletal muscle.
Assuntos
Mioblastos , Células Satélites de Músculo Esquelético , Animais , Diferenciação Celular , Conexinas/genética , Feminino , Masculino , Camundongos , Camundongos Knockout , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas , Músculo Esquelético , Proteínas do Tecido Nervoso/genéticaRESUMO
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.
Assuntos
Atrofia Muscular Espinal/genética , Distrofia Miotônica/genética , Proteínas de Ligação a RNA/genética , Processamento Alternativo/genética , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular Espinal/patologia , Distrofia Miotônica/patologia , Splicing de RNA/genética , RNA Mensageiro/genética , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Myotonic dystrophy type 1 (DM1) is a debilitating multisystemic disorder caused by a triplet repeat expansion in the 3' untranslated region of dystrophia myotonica protein kinase mRNAs. Mutant mRNAs accumulate in the nucleus of affected cells and misregulate RNA-binding proteins, thereby promoting characteristic missplicing events. However, little is known about the signaling pathways that may be affected in DM1. Here, we investigated the status of activated protein kinase (AMPK) signaling in DM1 skeletal muscle and found that the AMPK pathway is markedly repressed in a DM1 mouse model (human skeletal actin-long repeat, HSALR) and patient-derived DM1 myoblasts. Chronic pharmacological activation of AMPK signaling in DM1 mice with 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) has multiple beneficial effects on the DM1 phenotype. Indeed, a 6-week AICAR treatment of DM1 mice promoted expression of a slower, more oxidative phenotype, improved muscle histology and corrected several events associated with RNA toxicity. Importantly, AICAR also had a dose-dependent positive effect on the spliceopathy in patient-derived DM1 myoblasts. In separate experiments, we also show that chronic treatment of DM1 mice with resveratrol as well as voluntary wheel running also rescued missplicing events in muscle. Collectively, our findings demonstrate the therapeutic potential of chronic AMPK stimulation both physiologically and pharmacologically for DM1 patients.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Distrofia Miotônica/tratamento farmacológico , Proteínas Quinases/genética , Proteínas de Ligação a RNA/genética , Ribonucleotídeos/administração & dosagem , Quinases Proteína-Quinases Ativadas por AMP , Aminoimidazol Carboxamida/administração & dosagem , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Atividade Motora/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Mioblastos/efeitos dos fármacos , Distrofia Miotônica/genética , Distrofia Miotônica/fisiopatologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Resveratrol/administração & dosagem , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Myotonic Dystrophy type 1 (DM1) is caused by an expansion of CUG repeats in DMPK mRNAs. This mutation affects alternative splicing through misregulation of RNA-binding proteins. Amongst pre-mRNAs that are mis-spliced, several code for proteins involved in calcium homeostasis suggesting that calcium-handling and signaling are perturbed in DM1. Here, we analyzed expression of such proteins in DM1 mouse muscle. We found that the levels of several sarcoplasmic reticulum proteins (SERCA1, sarcolipin and calsequestrin) are altered, likely contributing to an imbalance in calcium homeostasis. We also observed that calcineurin (CnA) signaling is hyperactivated in DM1 muscle. Indeed, CnA expression and phosphatase activity are both markedly increased in DM1 muscle. Coherent with this, we found that activators of the CnA pathway (MLP, FHL1) are also elevated. Consequently, NFATc1 expression is increased in DM1 muscle and becomes relocalized to myonuclei, together with an up-regulation of its transcriptional targets (RCAN1.4 and myoglobin). Accordingly, DM1 mouse muscles display an increase in oxidative metabolism and fiber hypertrophy. To determine the functional consequences of this CnA hyperactivation, we administered cyclosporine A, an inhibitor of CnA, to DM1 mice. Muscles of treated DM1 mice showed an increase in CUGBP1 levels, and an exacerbation of key alternative splicing events associated with DM1. Finally, inhibition of CnA in cultured human DM1 myoblasts also resulted in a splicing exacerbation of the insulin receptor. Together, these findings show for the first time that calcium-CnA signaling is hyperactivated in DM1 muscle and that such hyperactivation represents a beneficial compensatory adaptation to the disease.
Assuntos
Calcineurina/metabolismo , Distrofia Miotônica/genética , Miotonina Proteína Quinase/genética , Processamento Alternativo , Animais , Antígenos CD , Calcineurina/genética , Cálcio/metabolismo , Sinalização do Cálcio , Técnicas de Cultura de Células , Modelos Animais de Doenças , Fibroblastos/metabolismo , Homeostase , Humanos , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Distrofia Miotônica/metabolismo , Miotonina Proteína Quinase/metabolismo , Fatores de Transcrição NFATC , Splicing de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA , Receptor de Insulina , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
Converging lines of evidence have now highlighted the key role for post-transcriptional regulation in the neuromuscular system. In particular, several RNA-binding proteins are known to be misregulated in neuromuscular disorders including myotonic dystrophy type 1, spinal muscular atrophy and amyotrophic lateral sclerosis. In this study, we focused on the RNA-binding protein Staufen1, which assumes multiple functions in both skeletal muscle and neurons. Given our previous work that showed a marked increase in Staufen1 expression in various physiological and pathological conditions including denervated muscle, in embryonic and undifferentiated skeletal muscle, in rhabdomyosarcomas as well as in myotonic dystrophy type 1 muscle samples from both mouse models and humans, we investigated the impact of sustained Staufen1 expression in postnatal skeletal muscle. To this end, we generated a skeletal muscle-specific transgenic mouse model using the muscle creatine kinase promoter to drive tissue-specific expression of Staufen1. We report that sustained Staufen1 expression in postnatal skeletal muscle causes a myopathy characterized by significant morphological and functional deficits. These deficits are accompanied by a marked increase in the expression of several atrophy-associated genes and by the negative regulation of PI3K/AKT signaling. We also uncovered that Staufen1 mediates PTEN expression through indirect transcriptional and direct post-transcriptional events thereby providing the first evidence for Staufen1-regulated PTEN expression. Collectively, our data demonstrate that Staufen1 is a novel atrophy-associated gene, and highlight its potential as a biomarker and therapeutic target for neuromuscular disorders and conditions.
Assuntos
Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Animais , Expressão Gênica , Camundongos , Camundongos Knockout , Denervação Muscular , Músculo Esquelético/metabolismo , Músculos/metabolismo , Atrofia Muscular/metabolismo , Atrofia Muscular Espinal/metabolismo , Distrofia Miotônica/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/genética , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Transdução de Sinais , TensinasRESUMO
Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by an expansion of CUG repeats in the 3' UTR of the DMPK gene. The CUG repeats form aggregates of mutant mRNA, which cause misregulation and/or sequestration of RNA-binding proteins, causing aberrant alternative splicing in cells. Previously, we showed that the multi-functional RNA-binding protein Staufen1 (Stau1) was increased in skeletal muscle of DM1 mouse models and patients. We also showed that Stau1 rescues the alternative splicing profile of pre-mRNAs, e.g. the INSR and CLC1, known to be aberrantly spliced in DM1. In order to explore further the potential of Stau1 as a therapeutic target for DM1, we first investigated the mechanism by which Stau1 regulates pre-mRNA alternative splicing. We report here that Stau1 regulates the alternative splicing of exon 11 of the human INSR via binding to Alu elements located in intron 10. Additionally, using a high-throughput RT-PCR screen, we have identified numerous Stau1-regulated alternative splicing events in both WT and DM1 myoblasts. A number of these aberrant ASEs in DM1, including INSR exon 11, are rescued by overexpression of Stau1. However, we find other ASEs in DM1 cells, where overexpression of Stau1 shifts the splicing patterns away from WT conditions. Moreover, we uncovered that Stau1-regulated ASEs harbour Alu elements in intronic regions flanking the alternative exon more than non-Stau1 targets. Taken together, these data highlight the broad impact of Stau1 as a splicing regulator and suggest that Stau1 may act as a disease modifier in DM1.
Assuntos
Processamento Alternativo/genética , Proteínas do Citoesqueleto/genética , Miotonina Proteína Quinase/genética , Proteínas de Ligação a RNA/genética , Expansão das Repetições de Trinucleotídeos/genética , Regiões 3' não Traduzidas , Elementos Alu/genética , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Proteínas do Citoesqueleto/metabolismo , Humanos , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mioblastos/metabolismo , Mioblastos/patologia , Distrofia Miotônica , Miotonina Proteína Quinase/metabolismo , Ligação Proteica , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismoRESUMO
Pannexin 1 (Panx1) and Pannexin 3 (Panx3) are single membrane channels recently implicated in myogenic commitment, as well as myoblast proliferation and differentiation in vitro. However, their expression patterns during skeletal muscle development and regeneration had yet to be investigated. Here, we show that Panx1 levels increase during skeletal muscle development becoming highly expressed together with Panx3 in adult skeletal muscle. In adult mice, Panx1 and Panx3 were differentially expressed in fast- and slow-twitch muscles. We also report that Panx1/PANX1 and Panx3/PANX3 are co-expressed in mouse and human satellite cells, which play crucial roles in skeletal muscle regeneration. Interestingly, Panx1 and Panx3 levels were modulated in muscle degeneration/regeneration, similar to the pattern seen during skeletal muscle development. As Duchenne muscular dystrophy is characterized by skeletal muscle degeneration and impaired regeneration, we next used mild and severe mouse models of this disease and found a significant dysregulation of Panx1 and Panx3 levels in dystrophic skeletal muscles. Together, our results are the first demonstration that Panx1 and Panx3 are differentially expressed amongst skeletal muscle types with their levels being highly modulated during skeletal muscle development, regeneration, and dystrophy. These findings suggest that Panx1 and Panx3 channels may play important and distinct roles in healthy and diseased skeletal muscles.
Assuntos
Conexinas/metabolismo , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Camundongos , Mioblastos/metabolismo , Regeneração/fisiologiaRESUMO
Background: Myotonic dystrophy type 1 (DM1) is a slowly progressive disease caused by abnormal CTG repetitions on the dystrophia myotonica protein kinase (DMPK) gene. Long mRNA from CTG repetitions stabilizes in nuclear foci and sequester muscleblind-like splicing regulator 1 (MBNL1). Cardinal signs of DM1 include muscle wasting and weakness. The impacts of DM1 progression on skeletal muscle are under-researched. Objective: Identifying physiopathological markers related to maximal strength loss over time in DM1. Methods: Twenty-two individuals with DM1 participated in two maximal isometric muscle strength (MIMS) evaluations of their knee extensors and two vastus lateralis muscle biopsies, 3 years apart. Muscle fiber typing, size (including minimal Feret's diameter [MFD] and atrophy/hypertrophy factors [AF/HF]), and nuclear foci and MBNL1 colocalization (foci/MBNL1+) were evaluated. Immunoblotting was used to measure glycogen synthase kinase-3 beta (GSK3ß), p62, LC3BI, LC3BII, and oxidative phosphorylation proteins. Results: There are significant correlations between the fold changes of MIMS with type 1 fiber MFD (ρ=â0.483) and AF (ρ=â-0.514). Regression analysis shows that baseline percentage of foci/MBNL1+ nuclei and strength training explain 44.1% of foci/MBNL1+ nuclei percentage variation over time. There are fair to excellent correlations between the fold changes of MIMS and GSK3ß (ρ=â0.327), p62 (ρ=â0.473), LC3BI (ρ=â0.518), LC3BII (ρ=â-0.391) and LC3BII/LC3BI (ρ=â-0.773). Conclusion: Type 1 MFD decrease and AF increase are correlated with MIMS loss. There seems to be a plateau effect in foci/MBNL1+ nuclei accumulation and strength training helps decrease this accumulation. Autophagy marker LC3BII/LC3BI ratio has a good biomarker potential of MIMS loss, but more investigations are needed.
RESUMO
Myotonic dystrophy type 1 (DM1) is a multisystemic disorder for which there is no cure. In recent years, progress has been made in defining disease mechanisms and in developing novel therapies, especially for skeletal muscle defects. Here, we highlight the potential of activating AMP-activated protein kinase (AMPK) with different approaches in combinatorial therapies.
Assuntos
Distrofia Miotônica , Humanos , Músculo Esquelético/metabolismo , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Distrofia Miotônica/terapiaRESUMO
The absence of dystrophin in Duchenne muscular dystrophy disrupts the dystrophin-associated glycoprotein complex resulting in skeletal muscle fiber fragility and atrophy, associated with fibrosis as well as microtubule and neuromuscular junction disorganization. The specific, non-conventional cytoplasmic histone deacetylase 6 (HDAC6) was recently shown to regulate acetylcholine receptor distribution and muscle atrophy. Here, we report that administration of the HDAC6 selective inhibitor tubastatin A to the Duchenne muscular dystrophy, mdx mouse model increases muscle strength, improves microtubule, neuromuscular junction, and dystrophin-associated glycoprotein complex organization, and reduces muscle atrophy and fibrosis. Interestingly, we found that the beneficial effects of HDAC6 inhibition involve the downregulation of transforming growth factor beta signaling. By increasing Smad3 acetylation in the cytoplasm, HDAC6 inhibition reduces Smad2/3 phosphorylation, nuclear translocation, and transcriptional activity. These findings provide in vivo evidence that Smad3 is a new target of HDAC6 and implicate HDAC6 as a potential therapeutic target in Duchenne muscular dystrophy.
Assuntos
Distrofina , Distrofia Muscular de Duchenne , Camundongos , Animais , Distrofina/genética , Distrofina/metabolismo , Camundongos Endogâmicos mdx , Desacetilase 6 de Histona/genética , Desacetilase 6 de Histona/metabolismo , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Acetilação , Fator de Crescimento Transformador beta/metabolismo , Músculo Esquelético/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/metabolismo , Fibrose , Fenótipo , Atrofia Muscular/patologia , Glicoproteínas/metabolismoRESUMO
PURPOSE: Recent work has highlighted the therapeutic potential of targeting autophagy to modulate cell survival in a variety of diseases including cancer. Recently, we found that the RNA-binding protein Staufen1 (STAU1) is highly expressed in alveolar rhabdomyosarcoma (ARMS) and that this abnormal expression promotes tumorigenesis. Here, we asked whether STAU1 is involved in the regulation of autophagy in ARMS cells. METHODS: We assessed the impact of STAU1 expression modulation in ARMS cell lines (RH30 and RH41), non-transformed skeletal muscle cells (C2C12) and STAU1-transgenic mice using complementary techniques. RESULTS: We found that STAU1 silencing reduces autophagy in the ARMS cell lines RH30 and RH41, while increasing their apoptosis. Mechanistically, this inhibitory effect was found to be caused by a direct negative impact of STAU1 depletion on the stability of Beclin-1 (BECN1) and ATG16L1 mRNAs, as well as by an indirect inhibition of JNK signaling via increased expression of Dual specificity phosphatase 8 (DUSP8). Pharmacological activation of JNK or expression silencing of DUSP8 was sufficient to restore autophagy in STAU1-depleted cells. By contrast, we found that STAU1 downregulation in non-transformed skeletal muscle cells activates autophagy in a mTOR-dependent manner, without promoting apoptosis. A similar effect was observed in skeletal muscles obtained from STAU1-overexpressing transgenic mice. CONCLUSIONS: Together, our data indicate an effect of STAU1 on autophagy regulation in ARMS cells and its differential role in non-transformed skeletal muscle cells. Our findings suggest a cancer-specific potential of targeting STAU1 for the treatment of ARMS.
Assuntos
Autofagia/genética , Proteínas do Citoesqueleto/genética , Perfilação da Expressão Gênica/métodos , Músculo Esquelético/metabolismo , Proteínas de Ligação a RNA/genética , Rabdomiossarcoma Alveolar/genética , Animais , Apoptose/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Regulação para Baixo/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Músculo Esquelético/citologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologia , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismoRESUMO
Microtubules (MTs) are known to be post-translationally modified at the neuromuscular junction (NMJ), hence increasing their stability. To date however, the function(s) of the dynamic MT network and its relative stability in the formation and maintenance of NMJs remain poorly described. Stabilization of the MT is dependent in part on its acetylation status, and HDAC6 is capable of reversing this post-translational modification. Here, we report that HDAC6 preferentially accumulates at NMJs and that it contributes to the organization and the stability of NMJs. Indeed, pharmacological inhibition of HDAC6 protects against MT disorganization and reduces the size of acetylcholine receptor (AChR) clusters. Moreover, the endogenous HDAC6 inhibitor paxillin interacts with HDAC6 in skeletal muscle cells, colocalizes with AChR aggregates, and regulates the formation of AChR. Our findings indicate that the focal insertion of AChRs into the postsynaptic membrane is regulated by stable MTs and highlight how an MT/HDAC6/paxillin axis participates in the regulation of AChR insertion and removal to control the structure of NMJs.
Assuntos
Desacetilase 6 de Histona/metabolismo , Microtúbulos/enzimologia , Fibras Musculares Esqueléticas/enzimologia , Junção Neuromuscular/enzimologia , Receptores Colinérgicos/metabolismo , Membranas Sinápticas/enzimologia , Tubulina (Proteína)/metabolismo , Acetilação , Animais , Linhagem Celular , Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/genética , Inibidores de Histona Desacetilases/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtúbulos/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Paxilina/metabolismo , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Membranas Sinápticas/efeitos dos fármacosRESUMO
Collagen Q (COLQ) is a specific collagen that anchors acetylcholinesterase (AChE) in the synaptic cleft of the neuromuscular junction. So far, no mutation has been identified in the ACHE human gene but over 50 different mutations in the COLQ gene are causative for a congenital myasthenic syndrome (CMS) with AChE deficiency. Mice deficient for COLQ mimic most of the functional deficit observed in CMS patients. At the molecular level, a striking consequence of the absence of COLQ is an increase in the levels of acetylcholine receptor (AChR) mRNAs and proteins in vivo and in vitro in murine skeletal muscle cells. Here, we decipher the mechanisms that drive AChR mRNA upregulation in cultured muscle cells deficient for COLQ. We show that the levels of AChR ß-subunit mRNAs are post-transcriptionally regulated by an increase in their stability. We demonstrate that this process results from an activation of p38 MAPK and the cytoplasmic translocation of the nuclear RNA-binding protein human antigen R (HuR) that interacts with the AU-rich element located within AChR ß-subunit transcripts. This HuR/AChR transcript interaction induces AChR ß-subunit mRNA stabilization and occurs at a specific stage of myogenic differentiation. In addition, pharmacological drugs that modulate p38 activity cause parallel modifications of HuR protein and AChR ß-subunit levels. Thus, our study provides new insights into the signaling pathways that are regulated by ColQ-deficiency and highlights for the first time a role for HuR and p38 in mRNA stability in a model of congenital myasthenic syndrome.
RESUMO
Rhabdomyosarcoma is the most common soft tissue sarcoma in children and young adults. Rhabdomyosarcomas are skeletal muscle-like tumours that typically arise in muscle beds, and express key myogenic regulatory factors. However, their developmental program remains blocked in the proliferative phase with cells unable to exit the cell cycle to fuse into myotubes. Recently, we uncovered a key role for the RNA-binding protein Staufen1 during myogenic differentiation through the regulation of c-myc translation. Given the known implication of c-myc in rhabdomyosarcoma, we hypothesized in the current work that Staufen1 controls rhabdomyosarcoma tumorigenesis. Here, we report for the first time the novel role of Staufen1 in cancer, specifically in rhabdomyosarcoma. We demonstrate that Staufen1 is markedly upregulated in human rhabdomyosarcoma tumours and cell lines as compared to normal skeletal muscle. Moreover, we show that Staufen1 promotes the tumorigenesis of embryonal and alveolar rhabdomyosarcoma subtypes both in cell culture and in animal models. Finally, our data demonstrate that Staufen1 has differential roles in embryonal versus alveolar rhabdomyosarcoma through the control of proliferative and apoptotic pathways, respectively. Together, these results provide the first evidence for Staufen1's direct implication in cancer biology. Accordingly, Staufen1 thus represents a novel target for the development of future therapeutic strategies for rhabdomyosarcoma.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas de Ligação a RNA/metabolismo , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Embrionário/metabolismo , Animais , Apoptose , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos SCID , Invasividade Neoplásica , Rabdomiossarcoma Alveolar/patologia , Rabdomiossarcoma Embrionário/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Myotonic dystrophy (DM1) is caused by an expansion of CUG repeats (CUG(exp)) in the DMPK mRNA 3'UTR. CUG(exp)-containing mRNAs become toxic to cells by misregulating RNA-binding proteins. Here we investigated the consequence of this RNA toxicity on the cellular stress response. We report that cell stress efficiently triggers formation of stress granules (SGs) in proliferating, quiescent, and differentiated muscle cells, as shown by the appearance of distinct cytoplasmic TIA-1- and DDX3-containing foci. We show that Staufen1 is also dynamically recruited into these granules. Moreover, we discovered that DM1 myoblasts fail to properly form SGs in response to arsenite. This blockage was not observed in DM1 fibroblasts, demonstrating a cell type-specific defect. DM1 myoblasts display increased expression and sequestration of toxic CUG(exp) mRNAs compared with fibroblasts. Of importance, down-regulation of Staufen1 in DM1 myoblasts rescues SG formation. Together our data show that Staufen1 participates in the inhibition of SG formation in DM1 myoblasts. These results reveal that DM1 muscle cells fail to properly respond to stress, thereby likely contributing to the complex pathogenesis of DM1.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Distrofia Miotônica/metabolismo , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo , Animais , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Citoplasma/metabolismo , Grânulos Citoplasmáticos/patologia , Regulação para Baixo , Humanos , Camundongos , Fibras Musculares Esqueléticas/patologia , Mioblastos/metabolismo , Mioblastos/patologia , Distrofia Miotônica/genética , RNA Mensageiro/metabolismo , Estresse Fisiológico/genéticaRESUMO
In a recent issue of PLOS Genetics, we reported that the double-stranded RNA-binding protein, Staufen1, functions as a disease modifier in the neuromuscular disorder Myotonic Dystrophy Type I (DM1). In this work, we demonstrated that Staufen1 regulates the alternative splicing of exon 11 of the human Insulin Receptor, a highly studied missplicing event in DM1, through Alu elements located in an intronic region. Furthermore, we found that Staufen1 overexpression regulates numerous alternative splicing events, potentially resulting in both positive and negative effects in DM1. Here, we discuss our major findings and speculate on the details of the mechanisms by which Staufen1 could regulate alternative splicing, in both normal and DM1 conditions. Finally, we highlight the importance of disease modifiers, such as Staufen1, in the DM1 pathology in order to understand the complex disease phenotype and for future development of new therapeutic strategies.