Bridging integrator 1 (Bin1) deficiency in zebrafish results in centronuclear myopathy

Human Molecular Genetics, Jul 2014

Autosomal recessive centronuclear myopathy (CNM2), caused by mutations in bridging integrator 1 (BIN1), is a mildly progressive neuromuscular disorder characterized by abnormally centralized myonuclei and muscle weakness. BIN1 is important for membrane sensing and remodeling in vitro in different cell types. However, to fully understand the biological roles of BIN1 in vivo and to answer critical questions concerning the muscle-specific function of BIN1 in vertebrates, robust small animal models are required. In this study, we create and characterize a novel zebrafish model of CNM2 using antisense morpholinos. Immunofluorescence and histopathological analyses of Bin1-deficient zebrafish skeletal muscle reveal structural defects commonly reported in human CNM2 biopsies. Live imaging of zebrafish embryos shows defective calcium release in bin1 morphants, linking the presence of abnormal triads to impairments in intracellular signaling. RNA-mediated rescue assays demonstrate that knockdown of zebrafish bin1 can reliably examine the pathogenicity of novel BIN1 mutations in vivo. Finally, our results strongly suggest that the phosphoinositide-binding domain of BIN1, present only in skeletal muscle isoforms, may be more critical for muscle maturation and maintenance than for early muscle development. Overall, our data support that BIN1 plays an important role in membrane tubulation and may promote skeletal muscle weakness in CNM2 by disrupting machinery necessary for excitation–contraction coupling in vertebrate organisms. The reproducible phenotype of Bin1-deficient zebrafish, together with the generalized advantages of the teleost system, makes this model readily adaptable to high-throughput screening strategies and may be used to identify therapies for CNM2 and related neuromuscular diseases.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

https://hmg.oxfordjournals.org/content/23/13/3566.full.pdf

Bridging integrator 1 (Bin1) deficiency in zebrafish results in centronuclear myopathy

Human Molecular Genetics Bridging integrator 1 (Bin1) deficiency in zebrafish results in centronuclear myopathy Laura L. Smith 0 Vandana A. Gupta 0 Alan H. Beggs 0 0 Division of Genetics and Genomics, The Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School , Boston, MA 02115 , USA Autosomal recessive centronuclear myopathy (CNM2), caused by mutations in bridging integrator 1 (BIN1), is a mildly progressive neuromuscular disorder characterized by abnormally centralized myonuclei and muscle weakness. BIN1 is important for membrane sensing and remodeling in vitro in different cell types. However, to fully understand the biological roles of BIN1 in vivo and to answer critical questions concerning the muscle-specific function of BIN1 in vertebrates, robust small animal models are required. In this study, we create and characterize a novel zebrafish model of CNM2 using antisense morpholinos. Immunofluorescence and histopathological analyses of Bin1-deficient zebrafish skeletal muscle reveal structural defects commonly reported in human CNM2 biopsies. Live imaging of zebrafish embryos shows defective calcium release in bin1 morphants, linking the presence of abnormal triads to impairments in intracellular signaling. RNA-mediated rescue assays demonstrate that knockdown of zebrafish bin1 can reliably examine the pathogenicity of novel BIN1 mutations in vivo. Finally, our results strongly suggest that the phosphoinositide-binding domain of BIN1, present only in skeletal muscle isoforms, may be more critical for muscle maturation and maintenance than for early muscle development. Overall, our data support that BIN1 plays an important role in membrane tubulation and may promote skeletal muscle weakness in CNM2 by disrupting machinery necessary for excitation - contraction coupling in vertebrate organisms. The reproducible phenotype of Bin1-deficient zebrafish, together with the generalized advantages of the teleost system, makes this model readily adaptable to high-throughput screening strategies and may be used to identify therapies for CNM2 and related neuromuscular diseases. INTRODUCTION Centronuclear myopathies (CNMs) are a heterogeneous group of congenital disorders characterized by muscle weakness and abnormal nuclear centralization in myofibers. Several genetic forms have now been described, and vary in terms of age of onset, severity of clinical symptoms and mode of inheritance. Pathogenic CNM mutations are most commonly reported in the phosphoinositide (PI) phosphatase myotubularin (MTM1) and the large GTPase dynamin 2 (DNM2) genes, resulting in neonatal X-linked (CNMX; MIM #310400) and autosomal dominant (CNM1; MIM #160150) forms of the disease, respectively ( 1,2 ). Variants in other genes, including the ryanodine receptor calcium release channel (RYR1), titin (TTN) and myotubularinrelated protein 14 (MTMR14), have also been identified in rare cases with centralized nuclei on muscle biopsy ( 3 – 6 ). Autosomal recessive CNM (CNM2; MIM #255200) is most often associated with homozygous partial loss-of-function mutations in the bridging integrator 1 (BIN1) gene ( 7 ). Compared with MTM1- and DNM2-related CNM, pathological mechanisms underlying BIN1-related CNM remain particularly unclear, owing to fewer available human biopsies as well as a lack of faithful and accessible animal models. BIN1 is a scaffolding protein involved in the tubular invagination of membranes and in membrane trafficking ( 8 – 10 ). Alternative splicing of the BIN1 gene generates at least 12 isoforms with diverse functions ( 11 – 14 ). All known protein isoforms possess an N-terminal amphipathic helix, a BAR (Bin/Amphiphysin/Rvs) domain able to sense and promote membrane curvature through dimerization and a C-terminal SH3 domain important for protein – protein interactions ( 15 – 17 ). Variability arises in the protein’s central region, where a clathrin-binding domain is present in neuronal isoforms and a PI-binding domain is found almost exclusively in skeletal muscle isoforms ( 11,13,18 ). Five of the six partial loss-of-function BIN1 mutations in CNM described to date are located in the ubiquitously expressed and evolutionarily conserved BAR and SH3 domains (Fig. 1D). BAR missense mutations strongly decrease BIN1 membrane tubulating properties in cultured cells, whereas SH3 truncating mutations impair interactions with DNM2 (7). Only recently was the first human BIN1 splice mutation affecting the muscle-specific PI-binding domain identified, and found to result in an unusually progressive form of the myopathy ( 19 ). The molecular basis of the muscle specificity of CNM2 remains largely unresolved. BIN1 expression increases dramatically during muscle cell growth and differentiation ( 12,20 ). In humans, the subcellular localization of BIN1 protein shifts during skeletal muscle development. BIN1 immunoreactivity appears as longitudinal striations along the muscle fiber in (...truncated)


This is a preview of a remote PDF: https://hmg.oxfordjournals.org/content/23/13/3566.full.pdf

Laura L. Smith, Vandana A. Gupta, Alan H. Beggs. Bridging integrator 1 (Bin1) deficiency in zebrafish results in centronuclear myopathy, Human Molecular Genetics, 2014, pp. 3566-3578, 23/13, DOI: 10.1093/hmg/ddu067