Regulation of DMD pathology by an ankyrin-encoded miRNA

<p>Abstract</p> <p>Background</p> <p>Duchenne muscular dystrophy (DMD) is an X-linked myopathy resulting from the production of a nonfunctional dystrophin protein. MicroRNA (miRNA) are small 21- to 24-nucleotide RNA that can regulate both individual genes and entire cell signaling pathways. Previously, we identified several mRNA, both muscle-enriched and inflammation-induced, that are dysregulated in the skeletal muscles of DMD patients. One particularly muscle-enriched miRNA, miR-486, is significantly downregulated in dystrophin-deficient mouse and human skeletal muscles. miR-486 is embedded within the <it>ANKYRIN1(ANK1) </it>gene locus, which is transcribed as either a long (erythroid-enriched) or a short (heart muscle- and skeletal muscle-enriched) isoform, depending on the cell and tissue types.</p> <p>Results</p> <p>Inhibition of miR-486 in normal muscle myoblasts results in inhibited migration and failure to repair a wound in primary myoblast cell cultures. Conversely, overexpression of miR-486 in primary myoblast cell cultures results in increased proliferation with no changes in cellular apoptosis. Using bioinformatics and miRNA reporter assays, we have identified platelet-derived growth factor receptor β, along with several other downstream targets of the phosphatase and tensin homolog deleted on chromosome 10/AKT (PTEN/AKT) pathway, as being modulated by miR-486. The generation of muscle-specific transgenic mice that overexpress miR-486 revealed that miR-486 alters the cell cycle kinetics of regenerated myofibers <it>in vivo</it>, as these mice had impaired muscle regeneration.</p> <p>Conclusions</p> <p>These studies demonstrate a link for miR-486 as a regulator of the PTEN/AKT pathway in dystrophin-deficient muscle and an important factor in the regulation of DMD muscle pathology.</p

Mutations in the Satellite Cell Gene MEGF10 Cause a Recessive Congenital Myopathy with Minicores

We ascertained a nuclear family in which three of four siblings were affected with an unclassified autosomal recessive myopathy characterized by severe weakness, respiratory impairment, scoliosis, joint contractures, and an unusual combination of dystrophic and myopathic features on muscle biopsy. Whole genome sequence from one affected subject was filtered using linkage data and variant databases. A single gene, MEGF10, contained nonsynonymous mutations that co-segregated with the phenotype. Affected subjects were compound heterozygous for missense mutations c.976T > C (p.C326R) and c.2320T > C (p.C774R). Screening the MEGF10 open reading frame in 190 patients with genetically unexplained myopathies revealed a heterozygous mutation, c.211C > T (p.R71W), in one additional subject with a similar clinical and histological presentation as the discovery family. All three mutations were absent from at least 645 genotyped unaffected control subjects. MEGF10 contains 17 atypical epidermal growth factor-like domains, each of which contains eight cysteine residues that likely form disulfide bonds. Both the p.C326R and p.C774R mutations alter one of these residues, which are completely conserved in vertebrates. Previous work showed that murine Megf10 is required for preserving the undifferentiated, proliferative potential of satellite cells, myogenic precursors that regenerate skeletal muscle in response to injury or disease. Here, knockdown of megf10 in zebrafish by four different morpholinos resulted in abnormal phenotypes including unhatched eggs, curved tails, impaired motility, and disorganized muscle tissue, corroborating the pathogenicity of the human mutations. Our data establish the importance of MEGF10 in human skeletal muscle and suggest satellite cell dysfunction as a novel myopathic mechanism

Zebrafish models for human FKRP muscular dystrophies

Various muscular dystrophies are associated with the defective glycosylation of α-dystroglycan and are known to result from mutations in genes encoding glycosyltransferases. Fukutin-related protein (FKRP) was identified as a homolog of fukutin, the defective protein in Fukuyama-type congenital muscular dystrophy (FCMD), that is thought to function as a glycosyltransferase. Mutations in FKRP have been linked to a variety of phenotypes including Walker–Warburg syndrome (WWS), limb girdle muscular dystrophy (LGMD) 2I and congenital muscular dystrophy 1C (MDC1C). Zebrafish are a useful animal model to reveal the mechanism of these diseases caused by mutations in FKRP gene. Downregulating FKRP expression in zebrafish by two different morpholinos resulted in embryos which had developmental defects similar to those observed in human muscular dystrophies associated with mutations in FKRP. The FKRP morphants showed phenotypes involving alterations in somitic structure and muscle fiber organization, as well as defects in developing eye morphology. Additionally, they were found to have a reduction in α-dystroglycan glycosylation and a shortened myofiber length. Moreover, co-injection of fish or human FKRP mRNA along with the morpholino restored normal development, α-dystroglycan glycosylation and laminin binding activity of α-dystroglycan in the morphants. Co-injection of the human FKRP mRNA containing causative mutations found in human patients of WWS, MDC1C and LGMD2I could not restore their phenotypes significantly. Interestingly, these morphant fish having human FKRP mutations showed a wide phenotypic range similar to that seen in humans

Characterization of Zebrafish Models of Marinesco-Sjögren Syndrome.

SIL1 is a nucleotide exchange factor for the endoplasmic reticulum chaperone, BiP. Mutations in the SIL1 gene cause Marinesco-Sjögren syndrome (MSS), an autosomal recessive disease characterized by cerebellar ataxia, mental retardation, congenital cataracts, and myopathy. To create novel zebrafish models of MSS for therapeutic drug screening, we analyzed phenotypes in sil1 knock down fish by two different antisense oligo morpholinos. Both sil1 morphants had abnormal formation of muscle fibers and irregularity of the myosepta. Moreover, they showed smaller-sized eyes and loss of purkinje cells in cerebellar area compared to controls. Immunoblotting analysis revealed increased protein amounts of BiP, lipidated LC3, and caspase 3. These data supported that the sil1 morphants can represent mimicking phenotypes of human MSS. The sil1 morphants phenocopy the human MSS disease pathology and are a good animal model for therapeutic studies

Protein expression analysis of sil1-morpholino injected fish.

<p>Western blot analysis of control morpholino (CMO) and sil1-morpholino2 injected fish (MO). The protein amounts of BiP, lipidated form of LC3 (LC3-II), and activated caspase 3 are significantly increased in <i>sil1</i> morphant embryos compared to those of CMO-injected embryos. A: Immunoblot with anti-BiP, LC3-II, activated-Caspase 3, and beta-actin. B: Relative expression level is analyzed via immunoblot (*p = 0.0226, ** p = 0.000102 and *** p = 0.000705, versus CMO, Student’s t-test, n = 3).</p

Staining of purkinje cells with anti-purvalbumin in cerebellar area.

<p>The number of purkinje cells detected with anti-parvalbumin shows reduced number of positive cells in MO1 or MO2 injected embryos. (A, B and C): CMO injected fish, (D, E and F): MO1 injected fish, (G, H and I): MO2 injected fish. (A, D and G: Staining Purkinje cell. (B, E and H): DAPI, nuclei. (C, F and I): Merged images. (J): Increased number of Purkinje cells in sil1 morphants with co-injection of fish sil1 mRNA (50 pg) from 23.1% (MO2, n = 13) to 64.3% (MO2+sil1 mRNA, n = 14).</p

Immunohistochemistry of skeletal muscle tissue of morpholino-injected fish.

<p>Immunostaining of CMO injected fish (A, B) and sil1 morphant (MO2) injected fish (C, D) with antibodies against beta-dystroglycan (beta-DG) (A, C) and myosin heavy chain (MHC) (B, D). Beta-dystroglycan expression at the myosepta of MO2 injected 4 dpf embryos is misshapen and has a less clear v-shaped structure. Staining with anti-MHC indicated that formation of myofibers is disturbed in MO2. Arrowheads indicate the disturbance of myosepta in MO2-injected fish. Bar: 100 μm. Arrows indicate the abnormal structure of myofibers.</p

RT-PCR and protein expression of sil1 morpholino-injected embryos.

<p>A: Morpholino oligo-nucleotides target for zebrafish sil1. Two different anti-sense morpholino oligo-nucleotides targeted to disrupt splicing of <i>sil1</i> mRNA are designed by Gene Tools LLC. B: A single RT-PCR product of 355 bp is seen in 4 dpf embryos injected 3 ng of control morpholino (CMO), whereas 4 dpf embryos injected 3 ng of sil1 morpholino 1 (MO1) had two different sized products of 355 and 446 bp. Two RT-PCR products of 355 bp and 407 bp were detected in 4 dpf embryos injected morphant 2 (MO2). C: Western blotting analysis shows reduced amounts of sil1 protein extracted from 20 each of MO1- or MO2-injected morphants compared to 4 dpf wild-type embryos (WT) and CMO. D: Injections of MO1 or MO2 are effective to reduce the expression of sil1 protein to 66.5% (MO1, light gray) and 46.9% (MO2, gray) compared to CMO-injected fish (black), respectively.</p