Expression of the neuropathy-associated MTMR2 gene rescues MTM1-associated myopathy.

Myotubularins (MTMs) are active or dead phosphoinositides phosphatases defining a large protein family conserved through evolution and implicated in different neuromuscular diseases. Loss-of-function mutations in MTM1 cause the severe congenital myopathy called myotubular myopathy (or X-linked centronuclear myopathy) while mutations in the MTM1-related protein MTMR2 cause a recessive Charcot-Marie-Tooth peripheral neuropathy. Here we aimed to determine the functional specificity and redundancy of MTM1 and MTMR2, and to assess their abilities to compensate for a potential therapeutic strategy. Using molecular investigations and heterologous expression of human MTMs in yeast cells and in Mtm1 knockout mice, we characterized several naturally occurring MTMR2 isoforms with different activities. We identified the N-terminal domain as responsible for functional differences between MTM1 and MTMR2. An N-terminal extension observed in MTMR2 is absent in MTM1, and only the short MTMR2 isoform lacking this N-terminal extension behaved similarly to MTM1 in yeast and mice. Moreover, adeno-associated virus-mediated exogenous expression of several MTMR2 isoforms ameliorates the myopathic phenotype owing to MTM1 loss, with increased muscle force, reduced myofiber atrophy, and reduction of the intracellular disorganization hallmarks associated with myotubular myopathy. Noteworthy, the short MTMR2 isoform provided a better rescue when compared with the long MTMR2 isoform. In conclusion, these results point to the molecular basis for MTMs functional specificity. They also provide the proof-of-concept that expression of the neuropathy-associated MTMR2 gene improves the MTM1-associated myopathy, thus identifying MTMR2 as a novel therapeutic target for myotubular myopathy.

A phosphoinositide conversion mechanism for exit from endosomes.

Phosphoinositides are a minor class of short-lived membrane phospholipids that serve crucial functions in cell physiology ranging from cell signalling and motility to their role as signposts of compartmental membrane identity. Phosphoinositide 4-phosphates such as phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are concentrated at the plasma membrane, on secretory organelles, and on lysosomes, whereas phosphoinositide 3-phosphates, most notably phosphatidylinositol 3-phosphate (PI(3)P), are a hallmark of the endosomal system. Directional membrane traffic between endosomal and secretory compartments, although inherently complex, therefore requires regulated phosphoinositide conversion. The molecular mechanism underlying this conversion of phosphoinositide identity during cargo exit from endosomes by exocytosis is unknown. Here we report that surface delivery of endosomal cargo requires hydrolysis of PI(3)P by the phosphatidylinositol 3-phosphatase MTM1, an enzyme whose loss of function leads to X-linked centronuclear myopathy (also called myotubular myopathy) in humans. Removal of endosomal PI(3)P by MTM1 is accompanied by phosphatidylinositol 4-kinase-2α (PI4K2α)-dependent generation of PI(4)P and recruitment of the exocyst tethering complex to enable membrane fusion. Our data establish a mechanism for phosphoinositide conversion from PI(3)P to PI(4)P at endosomes en route to the plasma membrane and suggest that defective phosphoinositide conversion at endosomes underlies X-linked centronuclear myopathy caused by mutation of MTM1 in humans.

Mendelian disorders of PI metabolizing enzymes.

More than twenty different genetic diseases have been described that are caused by mutations in phosphoinositide metabolizing enzymes, mostly in phosphoinositide phosphatases. Although generally ubiquitously expressed, mutations in these enzymes, which are mainly loss-of-function, result in tissue-restricted clinical manifestations through mechanisms that are not completely understood. Here we analyze selected disorders of phosphoinositide metabolism grouped according to the principle tissue affected: the nervous system, muscle, kidney, the osteoskeletal system, the eye, and the immune system. We will highlight what has been learnt so far from the study of these disorders about not only the cellular and molecular pathways that are involved or are governed by phosphoinositides, but also the many gaps that remain to be filled to gain a full understanding of the pathophysiological mechanisms underlying the clinical manifestations of this steadily growing class of diseases, most of which still remain orphan in terms of treatment. This article is part of a Special Issue entitled Phosphoinositides.

The PTEN and Myotubularin phosphoinositide 3-phosphatases: linking lipid signalling to human disease.

Two classes of lipid phosphatases selectively dephosphorylate the 3 position of the inositol ring of phosphoinositide signaling molecules: the PTEN and the Myotubularin families. PTEN dephosphorylates PtdIns(3,4,5)P(3), acting in direct opposition to the Class I PI3K enzymes in the regulation of cell growth, proliferation and polarity and is an important tumor suppressor. Although there are several PTEN-related proteins encoded by the human genome, none of these appear to fulfill the same functions. In contrast, the Myotubularins dephosphorylate both PtdIns(3)P and PtdIns(3,5)P(2), making them antagonists of the Class II and Class III PI 3-kinases and regulators of membrane traffic. Both phosphatase groups were originally identified through their causal mutation in human disease. Mutations in specific myotubularins result in myotubular myopathy and Charcot-Marie-Tooth peripheral neuropathy; and loss of PTEN function through mutation and other mechanisms is evident in as many as a third of all human tumors. This chapter will discuss these two classes of phosphatases, covering what is known about their biochemistry, their functions at the cellular and whole body level and their influence on human health.

The PtdIns3P phosphatase myotubularin is a cytoplasmic protein that also localizes to Rac1-inducible plasma membrane ruffles.

Myotubularin, the phosphatase mutated in X-linked myotubular myopathy, was shown to dephosphorylate phosphatidylinositol 3-monophosphate (PtdIns3P) and was also reported to interact with nuclear transcriptional regulators from the trithorax family. We have characterized a panel of specific antibodies and investigated the subcellular localization of myotubularin. Myotubularin is not detected in the nucleus, and localizes mostly as a dense cytoplasmic network. Overexpression of myotubularin does not detectably affect vesicle trafficking in the mammalian cells investigated, in contrast to previous observations in yeast models. Both mutation of a key aspartate residue of myotubularin and dominant activation of Rac1 GTPase lead to the recruitment of myotubularin to specific plasma membrane domains. Localization to Rac1-induced ruffles is dependent on the presence of a domain highly conserved in the myotubularin family (that we named RID). We thus propose that myotubularin may dephosphorylate a subpool of PtdIns3P (or another related substrate) at the plasma membrane.