Person:

Smith, Laura L.

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by mutations of the myotubularin gene, MTM1. Myotubularin belongs to a large family of conserved lipid phosphatases that include both catalytically active and inactive myotubularin-related proteins (i.e., “MTMRs”). Biochemically, catalytically inactive MTMRs have been shown to form heteroligomers with active members within the myotubularin family through protein-protein interactions. However, the pathophysiological significance of catalytically inactive MTMRs remains unknown in muscle. By in vitro as well as in vivo studies, we have identified that catalytically inactive myotubularin-related protein 12 (MTMR12) binds to myotubularin in skeletal muscle. Knockdown of the mtmr12 gene in zebrafish resulted in skeletal muscle defects and impaired motor function. Analysis of mtmr12 morphant fish showed pathological changes with central nucleation, disorganized Triads, myofiber hypotrophy and whorled membrane structures similar to those seen in X-linked myotubular myopathy. Biochemical studies showed that deficiency of MTMR12 results in reduced levels of myotubularin protein in zebrafish and mammalian C2C12 cells. Loss of myotubularin also resulted in reduction of MTMR12 protein in C2C12 cells, mice and humans. Moreover, XLMTM mutations within the myotubularin interaction domain disrupted binding to MTMR12 in cell culture. Analysis of human XLMTM patient myotubes showed that mutations that disrupt the interaction between myotubularin and MTMR12 proteins result in reduction of both myotubularin and MTMR12. These studies strongly support the concept that interactions between myotubularin and MTMR12 are required for the stability of their functional protein complex in normal skeletal muscles. This work highlights an important physiological function of catalytically inactive phosphatases in the pathophysiology of myotubular myopathy and suggests a novel therapeutic approach through identification of drugs that could stabilize the myotubularin-MTMR12 complex and hence ameliorate this disorder.

The congenital myopathies are a diverse group of inherited neuromuscular disorders that manifest as skeletal muscle weakness at birth or in infancy, and are classically defined by the predominant morphological features observed on muscle biopsy. The goals of this dissertation were to better understand the pathophysiology behind these devastating diseases and to identify new therapeutic approaches through the use of faithful vertebrate models.

Due to their proliferative capacity, transparency, and well-characterized genome, zebrafish represent a robust vertebrate model system to study muscle development. In the first part of this work, we created and characterized a novel zebrafish model of centronuclear myopathy using antisense morpholinos targeting the bridging integrator 1 (bin1) gene. Bin1 morphant skeletal muscles revealed structural defects reported in human biopsies, and live calcium imaging offered new mechanistic insights linking abnormal triads to impairments in intracellular signaling.

Later studies focused on two forms of core myopathy, and utilized stable zebrafish models to guide development of targeted and effective therapies. We began by using TALE nucleases to generate germ line mutations in the zebrafish selenoprotein N (sepn1) gene, and in doing so created the first vertebrate to accurately model human SEPN1-related myopathy (SEPN1-RM). Sepn1 zebrafish mutants exhibited morphological abnormalities, reduced contractile strength, and skeletal muscle “cores” under electron microscopy. We then showed that the sepn1 phenotype could be ameliorated by pharmacological inhibition of a thiol oxidase localized at the sarcoplasmic reticulum. These data served as the first in vivo evidence to indicate that reactive oxygen species significantly contribute to SEPN1-RM, and may do so by impairing calcium re-uptake following muscle contraction. Finally, we performed a medium-throughput chemical screen on the closely related relatively relaxed (ryr1b) zebrafish, and identified JAK-STAT cytokine signaling as a druggable molecular pathway relevant to these pathologies.

In summary, these studies increase our knowledge of the affected systems in both centronuclear and core myopathies, and provide strong in vivo support that these conditions arise from defects in skeletal muscle excitation-contraction coupling. This work also further establishes zebrafish-based small molecule screens as a powerful tool for lead compound identification and drug development in human genetic disease.