Genetic Biomarkers for ALS Disease in Transgenic SOD1G93A Mice

The pathophysiological mechanisms of both familial and sporadic Amyotrophic Lateral Sclerosis (ALS) are unknown, although growing evidence suggests that skeletal muscle tissue is a primary target of ALS toxicity. Skeletal muscle biopsies were performed on transgenic SOD1G93A mice, a mouse model of ALS, to determine genetic biomarkers of disease longevity. Mice were anesthetized with isoflurane, and three biopsy samples were obtained per animal at the three main stages of the disease. Transcriptional expression levels of seventeen genes, Ankrd1, Calm1, Col19a1, Fbxo32, Gsr, Impa1, Mef2c, Mt2, Myf5, Myod1, Myog, Nnt, Nogo A, Pax7, Rrad, Sln and Snx10, were tested in each muscle biopsy sample. Total RNA was extracted using TRIzol Reagent according to the manufacturer's protocol, and variations in gene expression were assayed by real-time PCR for all of the samples. The Pearson correlation coefficient was used to determine the linear correlation between transcriptional expression levels throughout disease progression and longevity. Consistent with the results obtained from total skeletal muscle of transgenic SOD1G93A mice and 74-day-old denervated mice, five genes (Mef2c, Gsr, Col19a1, Calm1 and Snx10) could be considered potential genetic biomarkers of longevity in transgenic SOD1G93A mice. These results are important because they may lead to the exploration of previously unexamined tissues in the search for new disease biomarkers and even to the application of these findings in human studies

Overexpression of Akt1 Enhances Adipogenesis and Leads to Lipoma Formation in Zebrafish

<div><h3>Background</h3><p>Obesity is a complex, multifactorial disorder influenced by the interaction of genetic, epigenetic, and environmental factors. Obesity increases the risk of contracting many chronic diseases or metabolic syndrome. Researchers have established several mammalian models of obesity to study its underlying mechanism. However, a lower vertebrate model for conveniently performing drug screening against obesity remains elusive. The specific aim of this study was to create a zebrafish obesity model by over expressing the insulin signaling hub of the <em>Akt1</em> gene.</p> <h3>Methodology/Principal Findings</h3><p><em>Skin oncogenic transformation screening shows that a stable zebrafish transgenic of Tg(krt4Hsa.myrAkt1</em>)^cy18 displays severely obese phenotypes at the adult stage. In Tg(<em>krt4:Hsa.myrAkt1</em>)^cy18, the expression of exogenous human constitutively active Akt1 (myrAkt1) can activate endogenous downstream targets of mTOR, GSK-3α/β, and 70S6K. During the embryonic to larval transitory phase, the specific over expression of myrAkt1 in skin can promote hypertrophic and hyperplastic growth. From 21 hour post-fertilization (hpf) onwards, myrAkt1 transgene was ectopically expressed in several mesenchymal derived tissues. This may be the result of the integration position effect. Tg(<em>krt4:Hsa.myrAkt1</em>)^cy18 caused a rapid increase of body weight, hyperplastic growth of adipocytes, abnormal accumulation of fat tissues, and blood glucose intolerance at the adult stage. Real-time RT-PCR analysis showed the majority of key genes on regulating adipogenesis, adipocytokine, and inflammation are highly upregulated in Tg(<em>krt4:Hsa.myrAkt1</em>)^cy18. In contrast, the myogenesis- and skeletogenesis-related gene transcripts are significantly downregulated in Tg(<em>krt4:Hsa.myrAkt1</em>)^cy18, suggesting that excess adipocyte differentiation occurs at the expense of other mesenchymal derived tissues.</p> <h3>Conclusion/Significance</h3><p>Collectively, the findings of this study provide direct evidence that Akt1 signaling plays an important role in balancing normal levels of fat tissue in vivo. The obese zebrafish examined in this study could be a new powerful model to screen novel drugs for the treatment of human obesity.</p> </div

DUX4c Is Up-Regulated in FSHD. It Induces the MYF5 Protein and Human Myoblast Proliferation

Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contractions of the D4Z4 repeat array in 4q35. We have previously identified a double homeobox gene (DUX4) within each D4Z4 unit that encodes a transcription factor expressed in FSHD but not control myoblasts. DUX4 and its target genes contribute to the global dysregulation of gene expression observed in FSHD. We have now characterized the homologous DUX4c gene mapped 42 kb centromeric of the D4Z4 repeat array. It encodes a 47-kDa protein with a double homeodomain identical to DUX4 but divergent in the carboxyl-terminal region. DUX4c was detected in primary myoblast extracts by Western blot with a specific antiserum, and was induced upon differentiation. The protein was increased about 2-fold in FSHD versus control myotubes but reached 2-10-fold induction in FSHD muscle biopsies. We have shown by Western blot and by a DNA-binding assay that DUX4c over-expression induced the MYF5 myogenic regulator and its DNA-binding activity. DUX4c might stabilize the MYF5 protein as we detected their interaction by co-immunoprecipitation. In keeping with the known role of Myf5 in myoblast accumulation during mouse muscle regeneration DUX4c over-expression activated proliferation of human primary myoblasts and inhibited their differentiation. Altogether, these results suggested that DUX4c could be involved in muscle regeneration and that changes in its expression could contribute to the FSHD pathology

Skeletal muscle wasting and renewal: a pivotal role of myokine IL-6

Adult skeletal tissue is composed of heterogeneous population of cells that constantly self-renew by means of a controlled process of activation and proliferation of tissue-resident stem cells named satellite cells. Many growth factors, cytokines and myokines produced by skeletal muscle cells play critical roles in local regulation of the inflammatory process and skeletal muscle regeneration during different pathological conditions. IL-6 is a pleiotropic cytokine released in large amount during infection, autoimmunity and cancer. Low levels of IL-6 can promote activation of satellite cells and myotube regeneration while chronically elevated production promote skeletal muscle wasting. These distinct effects may be explained by a crosstalk of the IL-6/IL-6 receptor and gp130 trans-signaling pathway that oppose to regenerative and anti-inflammatory of the classical IL-6 receptor signaling pathway. Here we discuss on potential therapeutic strategies using monoclonal antibodies to IL-6R for the treatment of skeletal muscle wasting and cachexia. We also highlight on the IL-6/JAK/STAT and FGF/p38αβ MAPK signaling pathways in satellite cell activation and the use of protein kinase inhibitors for tailoring and optimizing satellite cell proliferation during the skeletal muscle renewal. Future investigations on the roles of the IL-6 classical and trans-signaling pathways in both immune and non-immune cells in skeletal muscle tissue will provide new basis for therapeutic approaches to reverse atrophy and degeneration of skeletal muscles in cancer and inflammatory diseases

A role for the myogenic determination gene Myf5 in adult regenerative myogenesis

The myogenic determination genes Myf5, Myod and Mrf4 direct skeletal muscle cell fate prenatally. In adult myogenesis, Myod has been shown to regulate myoblast differentiation, however, our understanding of satellite cell regulation is incomplete since the roles of Myf5 and Mrf4 had not been clearly defined. Here we examine the function of Myf5 and Mrf4 in the adult using recently generated alleles. Mrf4 is not expressed in normal or Myf5 null satellite cells and myoblasts, therefore excluding a role for this determination gene in adult muscle progenitors. Skeletal muscles of adult Myf5 null mice exhibit a subtle progressive myopathy. Crucially, adult Myf5 null mice exhibit perturbed muscle regeneration with a significant increase in muscle fibre hypertrophy, delayed differentiation, adipocyte accumulation, and fibrosis after freeze-injury. Satellite cell numbers are not significantly altered in Myf5 null animals and they show a modest impaired proliferation under some conditions in vitro. Mice double mutant for Myf5 and Dystrophin were more severely affected than single mutants, with enhanced necrosis and regeneration. Therefore, we show that Myf5 is a regulator of regenerative myogenesis and homeostasis, with functions distinct from those of Myod and Mrf4. (C) 2007 Elsevier Inc. All rights reserve

Small-RNA sequencing identifies dynamic microRNA deregulation during skeletal muscle lineage progression

Abstract Skeletal muscle satellite cells are quiescent adult resident stem cells that activate, proliferate and differentiate to generate myofibres following injury. They harbour a robust proliferation potential and self-renewing capacity enabling lifelong muscle regeneration. Although several classes of microRNAs were shown to regulate adult myogenesis, systematic examination of stage-specific microRNAs during lineage progression from the quiescent state is lacking. Here we provide a genome-wide assessment of the expression of small RNAs during the quiescence/activation transition and differentiation by RNA-sequencing. We show that the majority of small RNAs present in quiescent, activated and differentiated muscle cells belong to the microRNA class. Furthermore, by comparing expression in distinct cell states, we report a massive and dynamic regulation of microRNAs, both in numbers and amplitude, highlighting their pivotal role in regulation of quiescence, activation and differentiation. We also identify a number of microRNAs with reliable and specific expression in quiescence including several maternally-expressed miRNAs generated at the imprinted Dlk1-Dio3 locus. Unexpectedly, the majority of class-switching miRNAs are associated with the quiescence/activation transition suggesting a poised program that is actively repressed. These data constitute a key resource for functional analyses of miRNAs in skeletal myogenesis, and more broadly, in the regulation of stem cell self-renewal and tissue homeostasis

Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration

International audienceDistinct cell populations with regenerative capacity have been reported to contribute to myofibres after skeletal muscle injury, including non-satellite cells as well as myogenic satellite cells. However, the relative contribution of these distinct cell types to skeletal muscle repair and homeostasis and the identity of adult muscle stem cells remain unknown. We generated a model for the conditional depletion of satellite cells by expressing a human diphtheria toxin receptor under control of the murine Pax7 locus. Intramuscular injection of diphtheria toxin during muscle homeostasis, or combined with muscle injury caused by myotoxins or exercise, led to a marked loss of muscle tissue and failure to regenerate skeletal muscle. Moreover, the muscle tissue became infiltrated by inflammatory cells and adipocytes. This localised loss of satellite cells was not compensated for endogenously by other cell types, but muscle regeneration was rescued after transplantation of adult Pax7(+) satellite cells alone. These findings indicate that other cell types with regenerative potential depend on the presence of the satellite cell population, and these observations have important implications for myopathic conditions and stem cell-based therapeutic approaches