The muscular dystrophies are a group of heterogeneous genetic diseases characterized by progressive degeneration and weakness of skeletal muscle. Since the discovery of the first muscular dystrophy ...gene encoding dystrophin, a large number of genes have been identified that are involved in various muscle-wasting and neuromuscular disorders. Human genetic studies complemented by animal model systems have substantially contributed to our understanding of the molecular pathomechanisms underlying muscle degeneration. Moreover, these studies have revealed distinct molecular and cellular mechanisms that link genetic mutations to diverse muscle wasting phenotypes.
The Pathogenesis and Therapy of Muscular Dystrophies Guiraud, Simon; Aartsma-Rus, Annemieke; Vieira, Natassia M ...
Annual review of genomics and human genetics,
08/2015, Letnik:
16, Številka:
1
Journal Article
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Current molecular genomic approaches to human genetic disorders have led to an explosion in the identification of the genes and their encoded proteins responsible for these disorders. The ...identification of the gene altered by mutations in Duchenne and Becker muscular dystrophy was one of the earliest examples of this paradigm. The nearly 30 years of research partly outlined here exemplifies the road that similar current gene discovery protocols will be expected to travel, albeit much more rapidly owing to improved diagnosis of genetic disorders and an understanding of the spectrum of mutations thought to cause them. The identification of the protein dystrophin has led to a new understanding of the muscle cell membrane and the proteins involved in membrane stability, as well as new candidate genes for additional forms of muscular dystrophy. Animal models identified with naturally occurring mutations and developed by genetic manipulation have furthered the understanding of disease progression and underlying pathology. The biochemistry and molecular analysis of patient samples have led to the different dystrophin-dependent and -independent therapies that are currently close to or in human clinical trials. The lessons learned from decades of research on dystrophin have benefited the field of human genetics.
Cell-surface markers for prospective isolation of stem cells from human skeletal muscle have been difficult to identify. Such markers would be powerful tools for studying satellite cell function ...during homeostasis and in pathogenesis of diseases such as muscular dystrophies. In this study, we show that the tetraspanin KAI/CD82 is an excellent marker for prospectively isolating stem cells from human fetal and adult skeletal muscle. Human CD82+ muscle cells robustly engraft into a mouse model of muscular dystrophy. shRNA knockdown of CD82 in myogenic cells reduces myoblast proliferation, suggesting it is functionally involved in muscle homeostasis. CD82 physically interacts with alpha7beta1 integrin (α7β1-ITG) and with α-sarcoglycan, a member of the Dystrophin-Associated Glycoprotein Complex (DAPC), both of which have been linked to muscular dystrophies. Consistently, CD82 expression is decreased in Duchenne muscular dystrophy patients. Together, these findings suggest that CD82 function may be important for muscle stem cell function in muscular disorders.
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•Tetraspanin CD82 is a marker for prospective isolation of human muscle stem cells•CD82 knockdown in myogenic cells decreases their proliferation•CD82 is in a protein complex with α7-integrin and α-sarcoglycan•CD82 expression is decreased in dystrophic human muscle stem cells
In this article, Alexander, Rozkalne, and colleagues describe the identification of CD82 as a prospective marker for human muscle stem cells. Knockdown of CD82 in myogenic cells reduces myoblast proliferation and CD82 expression is reduced in dystrophic muscle stem cells, suggesting a link with muscle disease.
Skeletal muscle possesses a strong ability to regenerate following injury, a fact that has been largely attributed to satellite cells. Satellite cells are skeletal muscle stem cells located beneath ...the basal lamina of the myofiber, and are the principal cellular source of growth and regeneration in skeletal muscle. MicroRNAs (miRNAs) play key roles in modulating several cellular processes by targeting multiple mRNAs that comprise a single or multiple signaling pathway. Several miRNAs have been shown to regulate satellite cell activity, such as miRNA-489, which functions to maintain satellite cells in a quiescent state. Although muscle-specific miRNAs have been identified, many of the molecular mechanisms that regulate myogenesis that are regulated by miRNAs still remain unknown. In this study, we have shown that miR-128a is highly expressed in brain and skeletal muscle, and increases during myoblast differentiation. MiR-128a was found to regulate the target genes involved in insulin signaling, which include Insr (insulin receptor), Irs1 (insulin receptor substrate 1) and Pik3r1 (phosphatidylinositol 3-kinases regulatory 1) at both the mRNA and protein level. Overexpression of miR-128a in myoblasts inhibited cell proliferation by targeting IRS1. By contrast, inhibition of miR-128a induced myotube maturation and myofiber hypertrophy in vitro and in vivo. Moreover, our results demonstrate that miR-128a expression levels are negatively controlled by tumor necrosis factor α (TNF-α). TNF-α promoted myoblast proliferation and myotube hypertrophy by facilitating IRS1/Akt signaling via a direct decrease of miR-128a expression in both myoblasts and myotubes. In summary, we demonstrate that miR-128a regulates myoblast proliferation and myotube hypertrophy, and provides a novel mechanism through which IRS1-dependent insulin signaling is regulated in skeletal muscle.
Zebrafish are a preferred vertebrate model for delineating genotype-phenotype relationships. One of the most studied features of zebrafish is their exceptional swimming ability. By 7 days ...postfertilization (dpf), zebrafish spend over two-thirds of their time engaged in spontaneous swimming activity and several months later they are capable of attaining some of the fastest swimming velocities relative to body length ever recorded in the laboratory. However, laboratory-assembled flumes capable of achieving the slow flow velocities characteristics of larvae as well as the relatively fast maximal velocities of adults have not been described in sufficient detail to allow easy replication. Here we describe an easily assembled, open-source zebrafish-scaled flume for assessing swimming performance. The flume uses two independent spherical-impeller pumps modulated by a microcontroller to achieve flow velocities ranging from 1 to 70 cm s-1. The microcontroller also monitors water temperature and flow velocity and sends these data to a personal computer for real-time display and storage. Incremental protocols for assessing maximal swimming speed (Umax) were developed, stored in custom software, and then uploaded to the microcontroller in order to assess performance of larval (14, 21, 28 dpf), juvenile (35, 42 dpf), and adult (8, 22 month) zebrafish. The flume had sufficient range and sensitivity to detect developmental changes in Umax of larvae and juveniles, an 18-24% faster Umax of adult males vs. females, and a 14-20% age-related reduction in Umax for the oldest zebrafish. Detailed information is provided to assemble and operate this low-cost, versatile, and reliable tool for assessing zebrafish swimming performance.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Patients with a combined immunodeficiency characterized by normal numbers but impaired function of T and B cells had a homozygous p.Tyr20His substitution in transferrin receptor 1 (TfR1), encoded by ...TFRC. The substitution disrupts the TfR1 internalization motif, resulting in defective receptor endocytosis and markedly increased TfR1 expression on the cell surface. Iron citrate rescued the lymphocyte defects, and expression of wild-type but not mutant TfR1 rescued impaired transferrin uptake in patient-derived fibroblasts. Tfrc(Y20H/Y20H) mice recapitulated the immunological defects of patients. Despite the critical role of TfR1 in erythrocyte development and function, patients had only mild anemia and only slightly increased TfR1 expression in erythroid precursors. We show that STEAP3, a metalloreductase expressed in erythroblasts, associates with TfR1 and partially rescues transferrin uptake in patient-derived fibroblasts, suggesting that STEAP3 may provide an accessory TfR1 endocytosis signal that spares patients from severe anemia. These findings demonstrate the importance of TfR1 in adaptive immunity.
Presenting features of inflammatory bowel disease (IBD) are non-specific. We hypothesized that mRNA profiles could (1) identify genes and pathways involved in disease pathogenesis; (2) identify a ...molecular signature that differentiates IBD from other conditions; (3) provide insight into systemic and colon-specific dysregulation through study of the concordance of the gene expression.
Children (8-18 years) were prospectively recruited at the time of diagnostic colonoscopy for possible IBD. We used transcriptome-wide mRNA profiling to study gene expression in colon biopsies and paired whole blood samples. Using blood mRNA measurements, we fit a regression model for disease state prediction that was validated in an independent test set of adult subjects (GSE3365).
Ninety-eight children were recruited 39 Crohn's disease, 18 ulcerative colitis, 2 IBDU, 39 non-IBD. There were 1,118 significantly differentially (IBD vs non-IBD) expressed genes in colon tissue, and 880 in blood. The direction of relative change in expression was concordant for 106/112 genes differentially expressed in both tissue types. The regression model from the blood mRNA measurements distinguished IBD vs non-IBD disease status in the independent test set with 80% accuracy using only 6 genes. The overlap of 5 immune and metabolic pathways in the two tissue types was significant (p<0.001).
Blood and colon tissue from patients with IBD share a common transcriptional profile dominated by immune and metabolic pathways. Our results suggest that peripheral blood expression levels of as few as 6 genes (IL7R, UBB, TXNIP, S100A8, ALAS2, and SLC2A3) may distinguish patients with IBD from non-IBD.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Autism Spectrum Disorders (ASD) is a spectrum of highly heritable neurodevelopmental disorders in which known mutations contribute to disease risk in 20% of cases. Here, we report the results of the ...largest blood transcriptome study to date that aims to identify differences in 170 ASD cases and 115 age/sex-matched controls and to evaluate the utility of gene expression profiling as a tool to aid in the diagnosis of ASD. The differentially expressed genes were enriched for the neurotrophin signaling, long-term potentiation/depression, and notch signaling pathways. We developed a 55-gene prediction model, using a cross-validation strategy, on a sample cohort of 66 male ASD cases and 33 age-matched male controls (P1). Subsequently, 104 ASD cases and 82 controls were recruited and used as a validation set (P2). This 55-gene expression signature achieved 68% classification accuracy with the validation cohort (area under the receiver operating characteristic curve (AUC): 0.70 95% confidence interval CI: 0.62-0.77). Not surprisingly, our prediction model that was built and trained with male samples performed well for males (AUC 0.73, 95% CI 0.65-0.82), but not for female samples (AUC 0.51, 95% CI 0.36-0.67). The 55-gene signature also performed robustly when the prediction model was trained with P2 male samples to classify P1 samples (AUC 0.69, 95% CI 0.58-0.80). Our result suggests that the use of blood expression profiling for ASD detection may be feasible. Further study is required to determine the age at which such a test should be deployed, and what genetic characteristics of ASD can be identified.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin, which results in dysfunctional signaling pathways within muscle. Previously, we identified microRNA-486 ...(miR-486) as a muscle-enriched microRNA that is markedly reduced in the muscles of dystrophin-deficient mice (Dmdmdx-5Cv mice) and in DMD patient muscles. Here, we determined that muscle-specific transgenic overexpression of miR-486 in muscle of Dmdmdx-5Cv mice results in reduced serum creatine kinase levels, improved sarcolemmal integrity, fewer centralized myonuclei, increased myofiber size, and improved muscle physiology and performance. Additionally, we identified dedicator of cytokinesis 3 (DOCK3) as a miR-486 target in skeletal muscle and determined that DOCK3 expression is induced in dystrophic muscles. DOCK3 overexpression in human myotubes modulated PTEN/AKT signaling, which regulates muscle hypertrophy and growth, and induced apoptosis. Furthermore, several components of the PTEN/AKT pathway were markedly modulated by miR-486 in dystrophin-deficient muscle. Skeletal muscle-specific miR-486 overexpression in Dmdmdx-5Cv animals decreased levels of DOCK3, reduced PTEN expression, and subsequently increased levels of phosphorylated AKT, which resulted in an overall beneficial effect. Together, these studies demonstrate that stable overexpression of miR-486 ameliorates the disease progression of dystrophin-deficient skeletal muscle.
Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy characterized by an asymmetric progressive weakness and wasting of the facial, shoulder and upper arm muscles, ...frequently accompanied by hearing loss and retinal vasculopathy. FSHD is an autosomal dominant disease linked to chromosome 4q35, but the causative gene remains controversial. DUX4 is a leading candidate gene as causative of FSHD. However, DUX4 expression is extremely low in FSHD muscle, and there is no DUX4 animal model that mirrors the pathology in human FSHD. Here, we show that the misexpression of very low levels of human DUX4 in zebrafish development recapitulates the phenotypes seen in human FSHD patients. Microinjection of small amounts of human full-length DUX4 (DUX4-fl) mRNA into fertilized zebrafish eggs caused asymmetric abnormalities such as less pigmentation of the eyes, altered morphology of ears, developmental abnormality of fin muscle, disorganization of facial musculature and/or degeneration of trunk muscle later in development. Moreover, DUX4-fl expression caused aberrant localization of myogenic cells marked with α-actin promoter-driven enhanced green fluorescent protein outside somite boundary, especially in head region. These abnormalities were rescued by coinjection of the short form of DUX4 (DUX4-s). Our results suggest that the misexpression of DUX4-fl, even at extremely low level, can recapitulate the phenotype observed in FSHD patients in a vertebrate model. These results strongly support the current hypothesis for a role of DUX4 in FSHD pathogenesis. We also propose that DUX4 expression during development is important for the pathogenesis of FSHD.
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