P.4.4 BIN1 deficiency in zebrafish results in centronuclear myopathy

Abstract

Centronuclear myopathies (CNM) are a heterogeneous group of congenital disorders characterized by skeletal muscle weakness and the abnormal centralization of myonuclei. Several genetically distinct forms of CNM have now been described based on age of onset, severity of clinical symptoms, and mode of inheritance. Autosomal recessive centronuclear myopathy (CNM2) presents as moderate to severe weakness at birth or in early childhood and is associated with mutations in the BIN1 gene. BIN1 encodes bridging integrator 1 (BIN1), a protein shown to contribute to membrane remodeling in vitro and to the organization of T-tubules in Drosophila. However, limited progress has been made in the study of BIN1 in vertebrates due to the perinatal lethality that results from the systemic ablation of Bin1 expression in mice. To better understand BIN1 function (s) in vivo and its role in disease pathogenesis, we first created a zebrafish model of CNM2 using morpholino antisense technology. BIN1-deficient zebrafish exhibit morphological abnormalities and significantly impaired motor function early in development. Histopathological changes, such as irregularly positioned nuclei and malformed triads, underlie these outward defects and are consistent with changes in the human disease. This model is also used to introduce a reliable assay for the in vivo characterization of novel BIN1 mutations, and to show that the BIN1 PI-binding domain is not required to rescue the BIN1-deficient phenotype. To screen chemical therapeutics for CNM2, we are now pursuing a targeted knockout of the zebrafish bin1 gene using TALE nucleases. Successful conclusion of these studies will increase general understanding of the basic biology of CNM2, the affected systems, and the mechanisms that lead to skeletal muscle weakness. Identification of small molecules that slow or prevent derangements of skeletal muscle will also set the stage for preclinical testing of new therapies for CNM and related disorders. Centronuclear myopathies (CNM) are a heterogeneous group of congenital disorders characterized by skeletal muscle weakness and the abnormal centralization of myonuclei. Several genetically distinct forms of CNM have now been described based on age of onset, severity of clinical symptoms, and mode of inheritance. Autosomal recessive centronuclear myopathy (CNM2) presents as moderate to severe weakness at birth or in early childhood and is associated with mutations in the BIN1 gene. BIN1 encodes bridging integrator 1 (BIN1), a protein shown to contribute to membrane remodeling in vitro and to the organization of T-tubules in Drosophila. However, limited progress has been made in the study of BIN1 in vertebrates due to the perinatal lethality that results from the systemic ablation of Bin1 expression in mice. To better understand BIN1 function (s) in vivo and its role in disease pathogenesis, we first created a zebrafish model of CNM2 using morpholino antisense technology. BIN1-deficient zebrafish exhibit morphological abnormalities and significantly impaired motor function early in development. Histopathological changes, such as irregularly positioned nuclei and malformed triads, underlie these outward defects and are consistent with changes in the human disease. This model is also used to introduce a reliable assay for the in vivo characterization of novel BIN1 mutations, and to show that the BIN1 PI-binding domain is not required to rescue the BIN1-deficient phenotype. To screen chemical therapeutics for CNM2, we are now pursuing a targeted knockout of the zebrafish bin1 gene using TALE nucleases. Successful conclusion of these studies will increase general understanding of the basic biology of CNM2, the affected systems, and the mechanisms that lead to skeletal muscle weakness. Identification of small molecules that slow or prevent derangements of skeletal muscle will also set the stage for preclinical testing of new therapies for CNM and related disorders.