Loss of Catalytically Inactive Lipid Phosphatase Myotubularin-related Protein 12 Impairs Myotubularin Stability and Promotes Centronuclear Myopathy in Zebrafish

Vandana A Gupta,Alan H Beggs,Leonela Amoasii,Nathaniel E Goldman,Ethan A Talbot,Karim Hnia,Junko Shimazu,Stacey R Gundry,Jessica E Mcintire,Laura L Smith,Jessica R Bass,Jocelyn Laporte,Gregory A Cox

doi:10.1371/journal.pgen.1003583

Abstract

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.

Highlights

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by mutations of the MTM1 gene that encodes myotubularin [1,2]
We show that catalytically inactive MTMR12 interacts with myotubularin in skeletal muscle
MTM1-GST protein pulled down MTMR12-B10 protein whereas no interaction was observed with the GST alone, suggesting myotubularin and MTMR12 interact with each other by direct protein-protein interactions (Figure 1A)

Summary

Introduction

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by mutations of the MTM1 gene that encodes myotubularin [1,2]. Affected males are born with severe generalized hypotonia and weakness of skeletal muscles with respiratory insufficiency. In majority of cases the disease is fatal within the first months of life, but a proportion of affected males survive into their teens or beyond yet are non ambulant and require ventilatory support. Affected muscle fibers exhibit hypotrophy with a large number of centrally placed nuclei in a high proportion of myofibers. XLMTM is considered a subtype of centronuclear myopathy (CNM) [3]. MTM1 encodes a 39phosphoinositide (PtdIns3P) lipid phosphatase that catalyzes the dephosphorylation of phosphatidylinositol-3-phosphate (PtdIns3P) and phosphatidylinositol-3,5-bisphosphate PtdIns(3,5P)2 [4,5,6,7]. Phosphoinositides (PIs) are critical for a variety of physiological processes, including cell proliferation, cell death, motility, cyto-

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