Starvation‐induced MTMR13 and RAB21 activity regulates VAMP8 to promote autophagosome–lysosome fusion

Autophagy, the process for recycling cytoplasm in the lysosome, depends on membrane trafficking. We previously identified Drosophila Sbf as a Rab21 guanine nucleotide exchange factor (GEF) that acts with Rab21 in endosomal trafficking. Here, we show that Sbf/MTMR13 and Rab21 have conserved functions required for starvation‐induced autophagy. Depletion of Sbf/MTMR13 or Rab21 blocked endolysosomal trafficking of VAMP8, a SNARE required for autophagosome–lysosome fusion. We show that starvation induces Sbf/MTMR13 GEF and RAB21 activity, as well as their induced binding to VAMP8 (or closest Drosophila homolog, Vamp7). MTMR13 is required for RAB21 activation, VAMP8 interaction and VAMP8 endolysosomal trafficking, defining a novel GEF‐Rab‐effector pathway. These results identify starvation‐responsive endosomal regulators and trafficking that tunes membrane demands with changing autophagy status.     

The phosphoinositide-3-phosphatase MTMR2 associates with MTMR13, a membrane-associated pseudophosphatase also mutated in type 4B Charcot-Marie-Tooth disease

Charcot-Marie-Tooth disease type 4B (CMT4B) is a severe, demyelinating peripheral neuropathy characterized by distinctive, focally folded myelin sheaths. CMT4B is caused by recessively inherited mutations in either myotubularin-related 2 (MTMR2) or MTMR13 (also called SET-binding factor 2). MTMR2 encodes a member of the myotubularin family of phosphoinositide-3-phosphatases, which dephosphorylate phosphatidylinositol 3-phosphate (PI(3)P) and bisphosphate PI(3,5)P2. MTMR13 encodes a large, uncharacterized member of the myotubularin family. The MTMR13 phosphatase domain is catalytically inactive because the essential Cys and Arg residues are absent. Given the genetic association of both MTMR2 and MTMR13 with CMT4B, we investigated the biochemical relationship between these two proteins. We found that the endogenous MTMR2 and MTMR13 proteins are associated in human embryonic kidney 293 cells. MTMR2-MTMR13 association is mediated by coiled-coil sequences present in each protein. We also examined the cellular localization of MTMR2 and MTMR13 using fluorescence microscopy and subcellular fractionation. We found that (i) MTMR13 is a predominantly membrane-associated protein; (ii) MTMR2 and MTMR13 cofractionate in both a light membrane fraction and a cytosolic fraction; and (iii) MTMR13 membrane association is mediated by the segment of the protein which contains the pseudophosphatase domain. This work, which describes the first cellular or biochemical investigation of the MTMR13 pseudophosphatase protein, suggests that MTMR13 functions in association with MTMR2. Loss of MTMR13 function in CMT4B2 patients may lead to alterations in MTMR2 function and subsequent alterations in 3-phosphoinositide signaling. Such a mechanism would explain the strikingly similar phenotypes of patients with recessive mutations in either MTMR2 or MTMR13.     

Myotubularin-Related (MTMR) Phospholipid Phosphatase Proteins in the Peripheral Nervous System

Myotubularin-related proteins (MTMRs) constitute a broad family of ubiquitously expressed phosphatases with 14 members in humans, of which eight are catalytically active phosphatases, while six are catalytically inactive. Active MTMRs possess 3-phosphatase activity toward both PtdIns3P and PtdIns(3, 5)P ₂ poliphosphoinositides (PPIn), suggesting an involvement in intracellular trafficking and membrane homeostasis. Among MTMRs, catalytically active MTMR2 and inactive MTMR13 have a nonredundant function in nerve. Loss of either MTMR2 or MTMR13 causes Charcot–Marie–Tooth type 4B1 and B2 neuropathy, respectively, characterized by demyelination and redundant loops of myelin known as myelin outfoldings. In Mtmr2-null mouse nerves, these aberrant foldings occur at 3–4 weeks after birth, a time when myelination is established, and Schwann cells are still elongating to reach the final internodal length. Moreover, Mtmr2-specific ablation in Schwann cells is both sufficient and necessary to provoke CMT4B1 with myelin outfoldings. MTMR2 phospholipid phosphatase might regulate intracellular trafficking events and membrane homeostasis in Schwann cells during postnatal nerve development. In this review, we will discuss recent findings on the MTMR family with a major focus on MTMR2 and MTMR13 and their putative role in Schwann cell biology.     

Sbf/MTMR13 coordinates PI(3)P and Rab21 regulation in endocytic control of cellular remodeling

Cells rely on the coordinated regulation of lipid phosphoinositides and Rab GTPases to define membrane compartment fates along distinct trafficking routes. The family of disease-related myotubularin (MTM) phosphoinositide phosphatases includes catalytically inactive members, or pseudophosphatases, with poorly understood functions. We found that Drosophila MTM pseudophosphatase Sbf coordinates both phosphatidylinositol 3-phosphate (PI(3)P) turnover and Rab21 GTPase activation in an endosomal pathway that controls macrophage remodeling. Sbf dynamically interacts with class II phosphatidylinositol 3-kinase and stably recruits Mtm to promote turnover of a PI(3)P subpool essential for endosomal trafficking. Sbf also functions as a guanine nucleotide exchange factor that promotes Rab21 GTPase activation associated with PI(3)P endosomes. Of importance, Sbf, Mtm, and Rab21 function together, along with Rab11-mediated endosomal trafficking, to control macrophage protrusion formation. This identifies Sbf as a critical coordinator of PI(3)P and Rab21 regulation, which specifies an endosomal pathway and cortical control.     

MTMR9 Increases MTMR6 Enzyme Activity, Stability, and Role in Apoptosis

Myotubularin-related protein 6 (MTMR6) is a catalytically active member of the myotubularin (MTM) family, which is composed of 14 proteins. Catalytically active myotubularins possess 3-phosphatase activity dephosphorylating phosphatidylinositol-3-phoshate and phosphatidylinositol-3,5-bisphosphate, and some members have been shown to form homomers or heteromeric complexes with catalytically inactive myotubularins. We demonstrate that human MTMR6 forms a heteromer with an enzymatically inactive member myotubularin-related protein 9 (MTMR9), both in vitro and in cells. MTMR9 increased the binding of MTMR6 to phospholipids without changing the lipid binding profile. MTMR9 increased the 3-phosphatase activity of MTMR6 up to 6-fold. We determined that MTMR6 is activated up to 28-fold in the presence of phosphatidylserine liposomes. Together, MTMR6 activity in the presence of MTMR9 and assayed in phosphatidylserine liposomes increased 84-fold. Moreover, the formation of this heteromer in cells resulted in increased protein levels of both MTMR6 and MTMR9, probably due to the inhibition of degradation of both proteins. Furthermore, co-expression of MTMR6 and MTMR9 decreased etoposide-induced apoptosis, whereas decreasing both MTMR6 and MTMR9 by RNA interference led to increased cell death in response to etoposide treatment when compared with that seen with RNA interference of MTMR6 alone. Thus, MTMR9 greatly enhances the functions of MTMR6.Myotubularin proteins are a family of 14 proteins with the canonical dual specificity protein tyrosine phosphatase active site CX₅R motif (1–3). Eight members of the myotubularin family possess catalytic activity, dephosphorylating phosphatidylinositol 3-phosphate (PtdIns-3-P)⁴ and phosphatidylinositol 3,5-bisphosphate (PtdIns-3,5-P₂) at the D-3 position, and six members are not catalytically active because they lack the conserved cysteine residue in the protein tyrosine phosphatase motif that is required for activity. Interest in this group of proteins originated from the genetic evidence linking myotubularin, the founding member of this family, to myotubular myopathy, an X-linked disorder characterized by severe hypotonia and generalized muscle weakness (4). Subsequently, mutations in MTMR2 and in its inactive binding partner MTMR13 were linked to a subset of Charcot-Marie-Tooth disease type 4B, a demyelinating neurodegenerative disorder (5, 6).Despite near identical substrate specificity, biochemical and genetic evidence supports the hypothesis that myotubularin proteins are not redundant and have unique functions within cells (2, 7–9). The mechanisms by which loss of function of myotubularin proteins produce diseases are not known. Current evidence supports the hypothesis that each myotubularin protein regulates a specific pool of PtdIns-3-P and/or PtdIns-3,5-P₂, which in turn regulates a variety of cellular functions. Differences in tissue expression and subcellular localization play a role in the specificity of different myotubularins (10–15).The functions of myotubularin proteins are altered by the formation of heteromers between catalytically active and inactive members of the family. The initial biochemical purification of MTM1 demonstrated the presence of MTM1 homodimers and MTM1-3-phosphatase adapter protein (3PAP) heteromers (16), which was later described as MTMR12 (15, 17). MTMR2 was found to form heteromers with MTMR5 (13) and MTMR13 (18), and MTMR7 formed heteromers with MTMR9 (19). In each case, a catalytically active myotubularin protein interacted with an inactive protein. Heteromerization generated two important effects: increased catalytic activity of the active component (13, 15, 19, 20) and targeting of the heteromer to specific subcellular locations (15). Mutations in the inactive member MTMR13 result in a similar phenotype in patients as the mutations in its catalytically active binding partner MTMR2, indicating an indispensable role for the catalytically inactive subunit (21).Myotubularin proteins can be grouped into subfamilies based on homology. Closely related MTMR6, MTMR7, and MTMR8 comprise such a subfamily. We have previously characterized the interaction between mouse MTMR7 and MTMR9 proteins (19). In this report, we characterize the interaction between human MTMR6 and MTMR9. MTMR6 and MTMR9 have been shown to form a heteromeric complex in mouse and Caenorhabditis elegans (19, 22). MTMR6 has been shown to inhibit the activity of a calcium-activated potassium channel (type KCa3.1) (23, 24). Two screening experiments implicate MTMR6 as a regulator of apoptosis. By RNA microarray analysis, increased MTMR6 expression was observed in B cell chronic lymphoid leukemia cells with increased resistance to irradiation-induced apoptosis (25), whereas in an RNA interference screen in HeLa cells, decreased MTMR6 expression promoted apoptosis (26).Here we show that MTMR6 interacts with MTMR9 in vitro and in human cells. This interaction increases the phospholipid binding and enzymatic activity of MTMR6 in vitro. Co-expression of either subunit in cells dramatically increased the protein levels of the individual binding partners, suggesting that heteromer formation increases the stability of the proteins. Finally, MTMR9 was found to potentiate the effects of MTMR6 on apoptosis.     

Receptor mediated endocytosis 8 is a novel PI(3)P binding protein regulated by myotubularin-related 2

Myotubularin related protein 2 (MTMR2) is a member of the myotubularin family of phosphoinositide lipid phosphatases. Although MTMR2 dephosphorylates the phosphoinositides PI(3)P and PI(3,5)P2, the phosphoinositide binding proteins that are regulated by MTMR2 are poorly characterized. In this study, phosphoinositide affinity chromatography coupled to mass spectrometry identified receptor mediated endocytosis 8 (RME-8) as a novel PI(3)P binding protein. RME-8 co-localized with the PI(3)P marker DsRed-FYVE, while the N-terminal region of RME-8 is required for PI(3)P and PI(3,5)P(2) binding in vitro. Depletion of PI(3)P by MTMR2 S58A or wortmannin treatment attenuated RME-8 endosomal localization and co-localization with EGFR on early endosomes. Our results suggest a model in which the localization of RME-8 to endosomal compartments is spatially mediated by PI(3)P binding and temporally regulated by MTMR2 activity.     

Genetic interaction between MTMR2 and FIG4 phospholipid phosphatases involved in Charcot-Marie-Tooth neuropathies

We previously reported that autosomal recessive demyelinating Charcot-Marie-Tooth (CMT) type 4B1 neuropathy with myelin outfoldings is caused by loss of MTMR2 (Myotubularin-related 2) in humans, and we created a faithful mouse model of the disease. MTMR2 dephosphorylates both PtdIns3P and PtdIns(3,5)P(2), thereby regulating membrane trafficking. However, the function of MTMR2 and the role of the MTMR2 phospholipid phosphatase activity in vivo in the nerve still remain to be assessed. Mutations in FIG4 are associated with CMT4J neuropathy characterized by both axonal and myelin damage in peripheral nerve. Loss of Fig4 function in the plt (pale tremor) mouse produces spongiform degeneration of the brain and peripheral neuropathy. Since FIG4 has a role in generation of PtdIns(3,5)P(2) and MTMR2 catalyzes its dephosphorylation, these two phosphatases might be expected to have opposite effects in the control of PtdIns(3,5)P(2) homeostasis and their mutations might have compensatory effects in vivo. To explore the role of the MTMR2 phospholipid phosphatase activity in vivo, we generated and characterized the Mtmr2/Fig4 double null mutant mice. Here we provide strong evidence that Mtmr2 and Fig4 functionally interact in both Schwann cells and neurons, and we reveal for the first time a role of Mtmr2 in neurons in vivo. Our results also suggest that imbalance of PtdIns(3,5)P(2) is at the basis of altered longitudinal myelin growth and of myelin outfolding formation. Reduction of Fig4 by null heterozygosity and downregulation of PIKfyve both rescue Mtmr2-null myelin outfoldings in vivo and in vitro.     

Endosomal targeting of the phosphoinositide 3-phosphatase MTMR2 is regulated by an N-terminal phosphorylation site

MTMR2 is a member of the myotubularin family of inositol lipid phosphatases, a large protein-tyrosine phosphatase subgroup that is conserved from yeast to humans. Furthermore, the peripheral neuromuscular disease Charcot-Marie Tooth disease type 4B has been attributed to mutations in the mtmr2 gene. Because the molecular mechanisms regulating MTMR2 have been poorly defined, we investigated whether reversible phosphorylation might regulate MTMR2 function. We used mass spectrometry-based methods to identify a high stoichiometry phosphorylation site on serine 58 of MTMR2. Phosphorylation at Ser(58), or a phosphomimetic S58E mutation, markedly decreased MTMR2 localization to endocytic vesicular structures. In contrast, a phosphorylation-deficient MTMR2 mutant (S58A) displayed constitutive localization to early endocytic structures. This localization pattern was accompanied by displacement of a PI(3)P-specific sensor protein and an increase in signal transduction pathways. Thus, MTMR2 phosphorylation is likely to be a critical mechanism by which MTMR2 access to its lipid substrate(s) is temporally and spatially regulated, thereby contributing to the control of downstream endosome maturation events.     

Modulation of Local PtdIns3P Levels by the PI Phosphatase MTMR3 Regulates Constitutive Autophagy

Autophagy is a catabolic process that delivers cytoplasmic material to the lysosome for degradation. The mechanisms regulating autophagosome formation and size remain unclear. Here, we show that autophagosome formation was triggered by the overexpression of a dominant‐negative inactive mutant of Myotubularin‐related phosphatase 3 (MTMR3). Mutant MTMR3 partially localized to autophagosomes, and PtdIns3P and two autophagy‐related PtdIns3P‐binding proteins, GFP‐DFCP1 and GFP‐WIPI‐1α (WIPI49/Atg18), accumulated at sites of autophagosome formation. Knock‐down of MTMR3 increased autophagosome formation, and overexpression of wild‐type MTMR3 led to significantly smaller nascent autophagosomes and a net reduction in autophagic activity. These results indicate that autophagy initiation depends on the balance between PI 3‐kinase and PI 3‐phosphatase activity. Local levels of PtdIns3P at the site of autophagosome formation determine autophagy initiation and the size of the autophagosome membrane structure.     

Cardiotoxin III suppresses hepatocyte growth factor–stimulated migration and invasion of MDA‐MB‐231 cells

The hepatocyte growth factor (HGF)/c‐Met signalling pathway is deregulated in most cancers and associated with a poor prognosis in breast cancer. Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity. In this study, we use HGF as an invasive inducer to investigate the effect of CTX III on MDA‐MB‐231 cells. When cells were treated with non‐toxic doses of CTX III, CTX III inhibited the HGF‐promoted cell migration and invasion. CTX III significantly suppressed the HGF‐induced c‐Met phosphorylation and downstream activation of phosphatidylinositol 3‐kinase (PI3k)/Akt and extracellular signal‐regulated kinase (ERK) 1/2. Additionally, CTX III similar to wortmannin (a PI3K inhibitor) and U0126 (an upstream kinase regulating ERK1/2 inhibitor) attenuated cell migration and invasion induced by HGF. This effect was paralleled by a significant reduction in phosphorylation of IκBα kinase and IκBα and nuclear translocation of nuclear factor κB (NF‐κB) as well as a reduction of matrix metalloproteinase‐9 (MMP‐9) activity. Furthermore, the c‐Met inhibitor PHA665752 inhibited HGF‐induced MMP‐9 expression, cell migration and invasion, as well as the activation of ERK1/2 and PI3K/Akt, suggesting that ERK1/2 and PI3K/Akt activation occurs downstream of c‐Met activation. Taken together, these findings suggest that CTX III inhibits the HGF‐induced invasion and migration of MDA‐MB‐231 cells via HGF/c‐Met‐dependent PI3K/Akt, ERK1/2 and NF‐κB signalling pathways, leading to the downregulation of MMP‐9 expression. Copyright © 2014 John Wiley & Sons, Ltd.     

Sequential actions of myotubularin lipid phosphatases regulate endosomal PI(3)P and growth factor receptor trafficking

Two different human diseases, X-linked myotubular myopathy and Charcot-Marie-Tooth disease, result from mutant MTM1 or MTMR2 lipid phosphatases. Although events involved in endosomal PI(3)P and PI(3,5)P(2) synthesis are well established and pivotal in receptor signaling and degradation, enzymes involved in phosphoinositide degradation and their roles in trafficking are incompletely characterized. Here, we dissect the functions of the MTM1 and MTMR2 myotubularins and establish how they contribute to endosomal PI(3)P homeostasis. By mimicking loss of function in disease through siRNA-mediated depletion of the myotubularins, excess PI(3)P accumulates on early (MTM1) and late (MTMR2) endosomes. Surprisingly, the increased PI(3)P blocks the egress of epidermal growth factor receptors from early or late endosomes, suggesting that the accumulation of signaling receptors in distinct endosomes may contribute to the unique disease etiologies when MTM1 or MTMR2 are mutant. We further demonstrate that direct myotubularin binding to the type III PI 3-kinase complex hVps34/hVps15 leads to phosphatase inactivation. The lipid kinase-phosphatase interaction also precludes interaction of the PI 3-kinase with Rab GTPase activators. Thus, unique molecular complexes control kinase and phosphatase activation and locally regulate PI(3)P on discrete endosome populations, thereby providing a molecular rationale for related human myo- and neuropathies.     

Crystal structure of a phosphoinositide phosphatase, MTMR2: insights into myotubular myopathy and Charcot-Marie-Tooth syndrome

Myotubularin-related proteins are a large subfamily of protein tyrosine phosphatases (PTPs) that dephosphorylate D3-phosphorylated inositol lipids. Mutations in members of the myotubularin family cause the human neuromuscular disorders myotubular myopathy and type 4B Charcot-Marie-Tooth syndrome. The crystal structure of a representative member of this family, MTMR2, reveals a phosphatase domain that is structurally unique among PTPs. A series of mutants are described that exhibit altered enzymatic activity and provide insight into the specificity of myotubularin phosphatases toward phosphoinositide substrates. The structure also reveals that the GRAM domain, found in myotubularin family phosphatases and predicted to occur in approximately 180 proteins, is part of a larger motif with a pleckstrin homology (PH) domain fold. Finally, the MTMR2 structure will serve as a model for other members of the myotubularin family and provide a framework for understanding the mechanism whereby mutations in these proteins lead to disease.     

Mutations in MTMR13, a new pseudophosphatase homologue of MTMR2 and Sbf1, in two families with an autosomal recessive demyelinating form of Charcot-Marie-Tooth disease associated with early-onset glaucoma

Charcot-Marie-Tooth disease (CMT) with autosomal recessive (AR) inheritance is a heterogeneous group of inherited motor and sensory neuropathies. In some families from Japan and Brazil, a demyelinating CMT, mainly characterized by the presence of myelin outfoldings on nerve biopsies, cosegregated as an autosomal recessive trait with early-onset glaucoma. We identified two such large consanguineous families from Tunisia and Morocco with ages at onset ranging from 2 to 15 years. We mapped this syndrome to chromosome 11p15, in a 4.6-cM region overlapping the locus for an isolated demyelinating ARCMT (CMT4B2). In these two families, we identified two different nonsense mutations in the myotubularin-related 13 gene, MTMR13. The MTMR protein family includes proteins with a phosphoinositide phosphatase activity, as well as proteins in which key catalytic residues are missing and that are thus called "pseudophosphatases." MTM1, the first identified member of this family, and MTMR2 are responsible for X-linked myotubular myopathy and Charcot-Marie-Tooth disease type 4B1, an isolated peripheral neuropathy with myelin outfoldings, respectively. Both encode active phosphatases. It is striking to note that mutations in MTMR13 also cause peripheral neuropathy with myelin outfoldings, although it belongs to a pseudophosphatase subgroup, since its closest homologue is MTMR5/Sbf1. This is the first human disease caused by mutation in a pseudophosphatase, emphasizing the important function of these putatively inactive enzymes. MTMR13 may be important for the development of both the peripheral nerves and the trabeculum meshwork, which permits the outflow of the aqueous humor. Both of these tissues have the same embryonic origin.     

Bone marrow‐derived mesenchymal stem cells enhance autophagy via PI3K/AKT signalling to reduce the severity of ischaemia/reperfusion‐induced lung injury

Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow‐derived mesenchymal stem cells (BM‐MSCs) have great potential for cell therapy. However, the mechanisms underlying the BM‐MSC activation of autophagy to provide a therapeutic effect in ischaemia/reperfusion‐induced lung injury (IRI) remain unclear. Thus, we investigate the activation of autophagy in IRI following transplantation with BM‐MSCs. Seventy mice were pre‐treated with BM‐MSCs before they underwent lung IRI surgery in vivo. Human pulmonary micro‐vascular endothelial cells (HPMVECs) were pre‐conditioned with BM‐MSCs by oxygen‐glucose deprivation/reoxygenation (OGD) in vitro. Expression markers for autophagy and the phosphoinositide 3‐kinase/protein kinase B (PI3K/Akt) signalling pathway were analysed. In IRI‐treated mice, administration of BM‐MSCs significantly attenuated lung injury and inflammation, and increased the level of autophagy. In OGD‐treated HPMVECs, co‐culture with BM‐MSCs attenuated endothelial permeability by decreasing the level of cell death and enhanced autophagic activation. Moreover, administration of BM‐MSCs decreased the level of PI3K class I and p‐Akt while the expression of PI3K class III was increased. Finally, BM‐MSCs‐induced autophagic activity was prevented using the inhibitor LY294002. Administration of BM‐MSCs attenuated lung injury by improving the autophagy level via the PI3K/Akt signalling pathway. These findings provide further understanding of the mechanisms related to BM‐MSCs and will help to develop new cell‐based therapeutic strategies in lung injury.     

PI3P phosphatase activity is required for autophagosome maturation and autolysosome formation

Autophagosome formation is promoted by the PI3 kinase complex and negatively regulated by myotubularin phosphatases, indicating that regulation of local phosphatidylinositol 3‐phosphate (PtdIns3P) levels is important for this early phase of autophagy. Here, we show that the Caenorhabditis elegans myotubularin phosphatase MTM‐3 catalyzes PtdIns3P turnover late in autophagy. MTM‐3 acts downstream of the ATG‐2/EPG‐6 complex and upstream of EPG‐5 to promote autophagosome maturation into autolysosomes. MTM‐3 is recruited to autophagosomes by PtdIns3P, and loss of MTM‐3 causes increased autophagic association of ATG‐18 in a PtdIns3P‐dependent manner. Our data reveal critical roles of PtdIns3P turnover in autophagosome maturation and/or autolysosome formation.     

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

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.     

Mal connects TLR2 to PI3Kinase activation and phagocyte polarization

The recognition of bacterial lipoproteins by toll‐like receptor (TLR) 2 is pivotal for inflammation initiation and control in many bacterial infections. TLR2‐dependent signalling is currently believed to essentially require both adaptor proteins MyD88 (m yeloid d ifferentiation primary response gene 88) and Mal/TIRAP (M yD88‐a dapter‐l ike/TI R‐domain‐containing a daptor p rotein). TLR2‐dependent, but MyD88‐independent responses have not been described yet. We report here on a novel‐signalling pathway downstream of TLR2, which does not adhere to the established model. On stimulation of the TLR2/6 heterodimer with diacylated bacterial lipoproteins, Mal directly interacts with the regulatory subunit of phosphoinositide 3‐kinase (PI3K), p85α, in an inducible fashion. The Mal–p85α interaction drives PI3K‐dependent phosphorylation of Akt, phosphatidylinositol(3,4,5)P3 (PIP₃) generation and macrophage polarization. MyD88 is not essential for PI3K activation and Akt phosphorylation; however, cooperates with Mal for PIP₃ formation and accumulation at the leading edge. In contrast to TLR2/6, TLR2/1 does not require Mal or MyD88 for Akt phosphorylation. Hence, Mal specifically connects TLR2/6 to PI3K activation, PIP₃ generation and macrophage polarization.     

Neuroprotective effects of astaxanthin against oxygen and glucose deprivation damage via the PI3K/Akt/GSK3β/Nrf2 signalling pathway in vitro

Astaxanthin (ATX), which is the most abundant flavonoid in propolis, has previously shown neuroprotective properties against cerebral ischaemia‐induced apoptosis. However, the mechanisms by which ATX mediates its therapeutic effects are unclear. At present, we explored the underlying mechanisms involved in the protective effects of ATX via the phosphoinositide 3‐kinase (PI3K)/Akt/glycogen synthase kinase 3 beta (GSK3β)/nuclear factor erythroid 2‐related factor 2 (Nrf2) signalling pathway in SH‐SY5Y cells. The PI3K/Akt inhibitor LY294002 and GSK3β inhibitor LiCl were employed in this study. Pre‐treatment with ATX for 24 hours significantly decreased the oxygen and glucose deprivation (OGD)‐induced viability loss, reduced the proportion of apoptosis and regulated OGD‐mediated reactive oxygen species (ROS) production. Furthermore, ATX suppressed OGD‐caused mitochondrial membrane potential and decomposition of caspase‐3 to cleaved caspase‐3, and heightened the B‐cell lymphoma 2 (Bcl‐2)/Bax ratio. PI3K/Akt/GSK3β/Nrf2 signalling pathway activation in SH‐SY5Y cells was verified by Western blot. ATX and LiCl treatment raised the protein levels of p‐Akt, p‐GSK3β, nucleus Nrf2 and haeme oxygenase 1 (HO‐1). However, these protein expression levels decreased by treatment of LY294002. The above in vitro data indicate that ATX can confer neuroprotection against OGD‐induced apoptosis via the PI3K/Akt/GSK3β/Nrf2 signalling pathway.