Nerve growth factor improves the muscle regeneration capacity of muscle stem cells in dystrophic muscle.

Researchers have attempted to use gene- and cell-based therapies to restore dystrophin and alleviate the muscle weakness that results from Duchenne muscular dystrophy (DMD). Our research group has isolated populations of muscle-derived stem cells (MDSCs) from the postnatal skeletal muscle of mice. In comparison with satellite cells, MDSCs display an improved transplantation capacity in dystrophic mdx muscle that we attribute to their ability to undergo long-term proliferation, self-renewal, and multipotent differentiation, including differentiation toward endothelial and neuronal lineages. Here we tested whether the use of nerve growth factor (NGF) improves the transplantation efficiency of MDSCs. We used two methods of in vitro NGF stimulation: retroviral transduction of MDSCs with a CL-NGF vector and direct stimulation of MDSCs with NGF protein. Neither method of NGF treatment changed the marker profile or proliferation behavior of the MDSCs, but direct stimulation with NGF protein significantly reduced the in vitro differentiation ability of the cells. NGF stimulation also significantly enhanced the engraftment efficiency of MDSCs transplanted within the dystrophic muscle of mdx mice, resulting in the regeneration of numerous dystrophin-positive muscle fibers. These findings highlight the importance of NGF as a modulatory molecule, the study of which will broaden our understanding of its biologic role in the regeneration and repair of skeletal muscle by musclederived cells

Dystrophin expression in muscle following gene transfer with a fully deleted ("Gutted") adenovirus is markedly improved by Trans-acting adenoviral gene products

Helper-dependent adenoviruses (HDAd) are Ad vectors lacking all or most viral genes. They hold great promise for gene therapy of diseases such as Duchenne muscular dystrophy (DMD), because they are less immunogenic than E1/E3-deleted Ad (first-generation Ad or FGAd) and can carry the full-length (Fl) dystrophin (dys) cDNA (12 kb). We have compared the transgene expression of a HDAd (HDAdCMVDysFl) and a FGAd (FGAdCMV-dys) in cell culture (HeLa, C2C12 myotubes) and in the muscle of mdx mice (the mouse model for DMD). Both vectors encoded dystrophin regulated by the same cytomegalovirus (CMV) promoter. We demonstrate that the amount of dystrophin expressed was significantly higher after gene transfer with FGAdCMV-dys compared to HDAdCMVDysFl both in vitro and in vivo. However, gene transfer with HDAdCMVDysFl in the presence of a FGAd resulted in a significant increase of dystrophin expression indicating that gene products synthesized by the FGAd increase, in trans, the amount of dystrophin produced. This enhancement occurred in cell culture and after gene transfer in the muscle of mdx mice and dystrophic golden retriever (GRMD) dogs, another animal model for DMD. The E4 region of Ad is required for the enhancement, because no increase of dystrophin expression from HDAdCMVDysFl was observed in the presence of an E1/E4-deleted Ad in vitro and in vivo. The characterization of these enhancing gene products followed by their inclusion into an HDAd may be required to produce sufficient dystrophin to mitigate the pathology of DMD by HDAd-mediated gene transfer

An official American thoracic society workshop report: Translational research in rare respiratory diseases

Rare respiratory diseases (RRDs) are a heterogeneous group of disorders that collectively represent a significant health care burden. In recent years, strong advocacy and policy initiatives have led to advances in the implementation of research and clinical care for rare diseases. The development of specialized centers and research networks has facilitated support for affected individuals as well as emerging programs in basic, translational, and clinical research. In selected RRDs, subsequent gains in knowledge have informed the development of targeted therapies and effective diagnostic tests, but many gaps persist. There was therefore a desire to identify the elements contributing to an effective translational research program in RRDs. To this end, a workshop was convened in October 2015 with a focus on the implementation of effective transnational research networks and collaborations aimed at developing novel diagnostic and therapeutic tools. Key elements included an emphasis on molecular pathogenesis, the continuing engagement of patient advocacy groups and policy makers, the effective use of preclinical models in the translational research pipeline, and the detailed phenotyping of patient cohorts. During the course of the workshop, current logistical and knowledge gapswere identified, and new solutions or opportunities were highlighted

Differential regulation of myofibrillar proteins in skeletal muscles of septic mice

Sepsis elicits skeletal muscle atrophy as a result of decreased total protein synthesis and/or increased total protein degradation. It is unknown how and whether sepsis differentially affects the expression of specific myofibrillar proteins in respiratory and limb muscles. In this study, we measured the effects of sepsis myofibrillar mRNAs and their corresponding protein levels in the diaphragm (DIA) and tibialis anterior (TA) muscles in a murine cecal ligation and perforation (CLP) model of sepsis. Male mice (C57/BL6j) underwent CLP-induced sepsis. Sham-operated mice were subjected to the same surgical procedures, except for CLP. Mice were euthanized 24, 48, or 96 h postsurgery. Transcript and protein levels of autophagy-related genes, ubiquitin E3 ligases, and several myofibrillar genes were quantified. Sepsis elicited transient fiber atrophy in the DIA and prolonged atrophy in the TA. Atrophy was coincident with increased autophagy and ubiquitin E3 ligase expression. Myosin heavy chain isoforms decreased at 24 h in the DIA and across the time-course in the TA, myosin light chain isoforms decreased across the time-course in both muscles, and troponins T and C as well as tropomyosin decreased after 24 and 48 h in both the DIA and TA. α-Actin and troponin I were unaffected by sepsis. Sepsis-induced decreases in myofibrillar protein levels coincided with decreased mRNA expressions of these proteins, suggesting that transcriptional inhibition is involved. We hypothesize that sepsis-induced muscle atrophy is mediated by decreased transcription and enhanced degradation of specific myofibrillar proteins, including myosin heavy and light chains, troponin C, troponin T, and tropomyosin. © 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society

Transient immunosuppression by FK506 permits a sustained high-level dystrophin expression after adenovirus-mediated dystrophin minigene transfer to skeletal muscles of adult dystrophic (mdx) mice

NRC publication: Ye

Characterization of Human Alpha-Dystrobrevin Isoforms in HL-60 Human Promyelocytic Leukemia Cells Undergoing Granulocytic Differentiation

The biochemical properties and spatial localization of the protein alpha-dystrobrevin and other isoforms were investigated in cells of the human promyelocytic leukemia line HL-60 granulocytic differentiation as induced by retinoic acid (RA). Alpha-dystrobrevin was detected both in the cytosol and the nuclei of these cells, and a short isoform (gamma-dystrobrevin) was modified by tyrosine phosphorylation soon after the onset of the RA-triggered differentiation. Varying patterns of distribution of alpha-dystrobrevin and its isoforms could be discerned in HL-60 promyelocytes, RA-differentiated mature granulocytes, and human neutrophils. Moreover, the gamma-dystrobrevin isoform was found in association with actin and myosin light chain. The results provide new information about potential involvement of alpha-dystrobrevin and its splice isoforms in signal transduction in myeloid cells during induction of granulocytic differentiation and/or at the commitment stage of differentiation or phagocytic cells

Utrophin Binds Laterally along Actin Filaments and Can Couple Costameric Actin with Sarcolemma When Overexpressed in Dystrophin-deficient Muscle

Dystrophin is widely thought to mechanically link the cortical cytoskeleton with the muscle sarcolemma. Although the dystrophin homolog utrophin can functionally compensate for dystrophin in mice, recent studies question whether utrophin can bind laterally along actin filaments and anchor filaments to the sarcolemma. Herein, we have expressed full-length recombinant utrophin and show that the purified protein is fully soluble with a native molecular weight and molecular dimensions indicative of monomers. We demonstrate that like dystrophin, utrophin can form an extensive lateral association with actin filaments and protect actin filaments from depolymerization in vitro. However, utrophin binds laterally along actin filaments through contribution of acidic spectrin-like repeats rather than the cluster of basic repeats used by dystrophin. We also show that the defective linkage between costameric actin filaments and the sarcolemma in dystrophin-deficient mdx muscle is rescued by overexpression of utrophin. Our results demonstrate that utrophin and dystrophin are functionally interchangeable actin binding proteins, but that the molecular epitopes important for filament binding differ between the two proteins. More generally, our results raise the possibility that spectrin-like repeats may enable some members of the plakin family of cytolinkers to laterally bind and stabilize actin filaments