MTM1-mediated production of phosphatidylinositol 5-phosphate fuels the formation of podosome-like protrusions regulating myoblast fusion.

Myogenesis is a multistep process that requires a spatiotemporal regulation of cell events resulting finally in myoblast fusion into multinucleated myotubes. Most major insights into the mechanisms underlying fusion seem to be conserved from insects to mammals and include the formation of podosome-like protrusions (PLPs) that exert a driving force toward the founder cell. However, the machinery that governs this process remains poorly understood. In this study, we demonstrate that MTM1 is the main enzyme responsible for the production of phosphatidylinositol 5-phosphate, which in turn fuels PI5P 4-kinase α to produce a minor and functional pool of phosphatidylinositol 4,5-bisphosphate that concentrates in PLPs containing the scaffolding protein Tks5, Dynamin-2, and the fusogenic protein Myomaker. Collectively, our data reveal a functional crosstalk between a PI-phosphatase and a PI-kinase in the regulation of PLP formation.

Dysregulation of myelin synthesis and actomyosin function underlies aberrant myelin in CMT4B1 neuropathy.

Charcot-Marie-Tooth type 4B1 (CMT4B1) is a severe autosomal recessive demyelinating neuropathy with childhood onset, caused by loss-of-function mutations in the myotubularin-related 2 (MTMR2) gene. MTMR2 is a ubiquitously expressed catalytically active 3-phosphatase, which in vitro dephosphorylates the 3-phosphoinositides PtdIns3P and PtdIns(3,5)P2, with a preference for PtdIns(3,5)P2 A hallmark of CMT4B1 neuropathy are redundant loops of myelin in the nerve termed myelin outfoldings, which can be considered the consequence of altered growth of myelinated fibers during postnatal development. How MTMR2 loss and the resulting imbalance of 3'-phosphoinositides cause CMT4B1 is unknown. Here we show that MTMR2 by regulating PtdIns(3,5)P2 levels coordinates mTORC1-dependent myelin synthesis and RhoA/myosin II-dependent cytoskeletal dynamics to promote myelin membrane expansion and longitudinal myelin growth. Consistent with this, pharmacological inhibition of PtdIns(3,5)P2 synthesis or mTORC1/RhoA signaling ameliorates CMT4B1 phenotypes. Our data reveal a crucial role for MTMR2-regulated lipid turnover to titrate mTORC1 and RhoA signaling thereby controlling myelin growth.

Class III PI3K Biology.

Highly conserved from yeast to mammals, vacuolar protein sorting 34 (Vps34) is the sole member of the third class of the phosphoinositide 3-kinase (PI3K) family. By producing phosphatidylinositol-3-monophosphate (PtdIns3P) through its scaffolding function essential for the catalytic lipid activity, Vps34 regulates endosomal trafficking, autophagy, phagocytosis, and nutrient-sensing signaling. The development of genetically modified mouse models and specific inhibitors has largely contributed over the past ten years to a better understanding of Vps34 functions in biological and physiological processes in mammals and, ultimately, its potential implications and targeting in human diseases. This chapter will summarize the current knowledge of the structure and regulation of Vps34 as well as its cellular and organismal functions.

PI3KC2ß inactivation stabilizes VE-cadherin junctions and preserves vascular integrity.

Endothelium protection is critical, because of the impact of vascular leakage and edema on pathological conditions such as brain ischemia. Whereas deficiency of class II phosphoinositide 3-kinase alpha (PI3KC2α) results in an increase in vascular permeability, we uncover a crucial role of the beta isoform (PI3KC2ß) in the loss of endothelial barrier integrity following injury. Here, we studied the role of PI3KC2ß in endothelial permeability and endosomal trafficking in vitro and in vivo in ischemic stroke. Mice with inactive PI3KC2ß showed protection against vascular permeability, edema, cerebral infarction, and deleterious inflammatory response. Loss of PI3KC2ß in human cerebral microvascular endothelial cells stabilized homotypic cell-cell junctions by increasing Rab11-dependent VE-cadherin recycling. These results identify PI3KC2ß as a potential new therapeutic target to prevent aggravating lesions following ischemic stroke.

PIKfyve-Dependent Phosphoinositide Dynamics in Megakaryocyte/Platelet Granule Integrity and Platelet Functions.

BACKGROUND: Secretory granules are key elements for platelet functions. Their biogenesis and integrity are regulated by fine-tuned mechanisms that need to be fully characterized. Here, we investigated the role of the phosphoinositide 5-kinase PIKfyve and its lipid products, PtdIns5P (phosphatidylinositol 5 monophosphate) and PtdIns(3,5)P2 (phosphatidylinositol (3,5) bisphosphate) in granule homeostasis in megakaryocytes and platelets. METHODS: For that, we invalidated PIKfyve by pharmacological inhibition or gene silencing in megakaryocytic cell models (human MEG-01 cell line, human imMKCLs, mouse primary megakaryocytes) and in human platelets. RESULTS: We unveiled that PIKfyve expression and its lipid product levels increased with megakaryocytic maturation. In megakaryocytes, PtdIns5P and PtdIns(3,5)P2 were found in alpha and dense granule membranes with higher levels in dense granules. Pharmacological inhibition or knock-down of PIKfyve in megakaryocytes decreased PtdIns5P and PtdIns(3,5)P2 synthesis and induced a vacuolar phenotype with a loss of alpha and dense granule identity. Permeant PtdIns5P and PtdIns(3,5)P2 and the cation channel TRPML (transient receptor potential mucolipin) 1 and TPC (two pore segment channel) 2 activation were able to accelerate alpha and dense granule integrity recovery following release of PIKfyve pharmacological inhibition. In platelets, PIKfyve inhibition specifically impaired the integrity of dense granules culminating in defects in their secretion, platelet aggregation, and thrombus formation. CONCLUSIONS: These data demonstrated that PIKfyve and its lipid products PtdIns5P and PtdIns(3,5)P2 control granule integrity both in megakaryocytes and platelets.

What can we learn from the platelet lipidome?

Besides their proteome, platelets use, in all responses to the environmental cues, a huge and diverse family of hydrophobic and amphipathic small molecules involved in structural, metabolic and signaling functions; the lipids. Studying how platelet lipidome changes modulate platelet function is an old story constantly renewed through the impressive technical advances allowing the discovery of new lipids, functions and metabolic pathways. Technical progress in analytical lipidomic profiling by top-of-the-line approaches such as nuclear magnetic resonance and gas chromatography or liquid chromatography coupled to mass spectrometry enables either large-scale analysis of lipids or targeted lipidomics. With the support of bioinformatics tools and databases, it is now possible to investigate thousands of lipids over a concentration range of several orders of magnitude. The lipidomic landscape of platelets is considered a treasure trove, not only able to expand our knowledge of platelet biology and pathologies but also to bring diagnostic and therapeutic opportunities. The aim of this commentary article is to summarize the advances in the field and to highlight what lipidomics can tell us about platelet biology and pathophysiology.

What is the context? Lipids are a huge and diverse family of molecules strongly involved in biological membranes organization and dynamics, signal transduction, cell metabolism and intercellular communication.Earlier seminal works using conventional lipid biochemistry methods have shown the essential role of certain classes of lipids in platelet biology and platelet-related pathologiesWhat is new? The important development of modern lipidomic analyses using mass-spectrometry now provides opportunities to investigate the entire platelet lipidome in different conditions.The application of lipidomic approaches to analyze large-scale lipid species allows platelet clinical lipidomics development.What is the impact? Study of the lipidomic landscape of platelets will expand our knowledge of platelet biology and should bring new diagnosis and therapeutic opportunities.Evaluating the functional and clinical significance of the data generated by modern platelet lipidomics appears as a vast and exciting challenge.

SHIP1 Controls Internal Platelet Contraction and αIIbß3 Integrin Dynamics in Early Platelet Activation.

The Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) is known to dephosphorylate PtdIns(3,4,5)P3 into PtdIns(3,4)P2 and to interact with several signaling proteins though its docking functions. It has been shown to negatively regulate platelet adhesion and spreading on a fibrinogen surface and to positively regulate thrombus growth. In the present study, we have investigated its role during the early phase of platelet activation. Using confocal-based morphometric analysis, we found that SHIP1 is involved in the regulation of cytoskeletal organization and internal contractile activity in thrombin-activated platelets. The absence of SHIP1 has no significant impact on thrombin-induced Akt or Erk1/2 activation, but it selectively affects the RhoA/Rho-kinase pathway and myosin IIA relocalization to the cytoskeleton. SHIP1 interacts with the spectrin-based membrane skeleton, and its absence induces a loss of sustained association of integrins to this network together with a decrease in αIIbß3 integrin clustering following thrombin stimulation. This αIIbß3 integrin dynamics requires the contractile cytoskeleton under the control of SHIP1. RhoA activation, internal platelet contraction, and membrane skeleton integrin association were insensitive to the inhibition of PtdIns(3,4,5)P3 synthesis or SHIP1 phosphatase activity, indicating a role for the docking properties of SHIP1 in these processes. Altogether, our data reveal a lipid-independent function for SHIP1 in the regulation of the contractile cytoskeleton and integrin dynamics in platelets.

X-linked myotubular myopathy is associated with epigenetic alterations and is ameliorated by HDAC inhibition.

X-linked myotubular myopathy (XLMTM) is a fatal neuromuscular disorder caused by loss of function mutations in MTM1. At present, there are no directed therapies for XLMTM, and incomplete understanding of disease pathomechanisms. To address these knowledge gaps, we performed a drug screen in mtm1 mutant zebrafish and identified four positive hits, including valproic acid, which functions as a potent suppressor of the mtm1 zebrafish phenotype via HDAC inhibition. We translated these findings to a mouse XLMTM model, and showed that valproic acid ameliorates the murine phenotype. These observations led us to interrogate the epigenome in Mtm1 knockout mice; we found increased DNA methylation, which is normalized with valproic acid, and likely mediated through aberrant 1-carbon metabolism. Finally, we made the unexpected observation that XLMTM patients share a distinct DNA methylation signature, suggesting that epigenetic alteration is a conserved disease feature amenable to therapeutic intervention.

Platelet Lipidome Fingerprint: New Assistance to Characterize Platelet Dysfunction in Obesity.

Obesity is associated with a pro-inflammatory and pro-thrombotic state that supports atherosclerosis progression and platelet hyper-reactivity. During the last decade, the platelet lipidome has been considered a treasure trove, as it is a source of biomarkers for preventing and treating different pathologies. The goal of the present study was to determine the lipid profile of platelets from non-diabetic, severely obese patients compared with their age- and sex-matched lean controls. Lipids from washed platelets were isolated and major phospholipids, sphingolipids and neutral lipids were analyzed either by gas chromatography or by liquid chromatography coupled to mass spectrometry. Despite a significant increase in obese patient's plasma triglycerides, there were no significant differences in the levels of triglycerides in platelets among the two groups. In contrast, total platelet cholesterol was significantly decreased in the obese group. The profiling of phospholipids showed that phosphatidylcholine and phosphatidylethanolamine contents were significantly reduced in platelets from obese patients. On the other hand, no significant differences were found in the sphingomyelin and ceramide levels, although there was also a tendency for reduced levels in the obese group. The outline of the glycerophospholipid and sphingolipid molecular species (fatty-acyl profiles) was similar in the two groups. In summary, these lipidomics data indicate that platelets from obese patients have a unique lipid fingerprint that may guide further studies and provide mechanistic-driven perspectives related to the hyperactivate state of platelets in obesity.

Glycerophosphodiesterase 3 (GDE3) is a lysophosphatidylinositol-specific ectophospholipase C acting as an endocannabinoid signaling switch.

Endocannabinoid signaling plays a regulatory role in various (neuro)biological functions. 2-arachidonoylglycerol (2-AG) is the most abundant endocannabinoid, and although its canonical biosynthetic pathway involving phosphoinositide-specific phospholipase C and diacylglycerol lipase α is known, alternative pathways remain unsettled. Here, we characterize a noncanonical pathway implicating glycerophosphodiesterase 3 (GDE3, from GDPD2 gene). Human GDE3 expressed in HEK293T cell membranes catalyzed the conversion of lysophosphatidylinositol (LPI) into monoacylglycerol and inositol-1-phosphate. The enzyme was equally active against 1-acyl and 2-acyl LPI. When using 2-acyl LPI, where arachidonic acid is the predominant fatty acid, LC-MS analysis identified 2-AG as the main product of LPI hydrolysis by GDE3. Furthermore, inositol-1-phosphate release into the medium occurred upon addition of LPI to intact cells, suggesting that GDE3 is actually an ecto-lysophospholipase C. In cells expressing G-protein-coupled receptor GPR55, GDE3 abolished 1-acyl LPI-induced signaling. In contrast, upon simultaneous ex-pression of GDE3 and cannabinoid receptor CB2, 2-acyl LPI evoked the same signal as that induced by 2-AG. These data strongly suggest that, in addition to degrading the GPR55 LPI ligand, GDE3 can act as a switch between GPR55 and CB2 signaling. Coincident with a major expression of both GDE3 and CB2 in the spleen, spleens from transgenic mice lacking GDE3 displayed doubling of LPI content compared with WT mice. Decreased production of 2-AG in whole spleen was also observed, supporting the in vivo relevance of our findings. These data thus open a new research avenue in the field of endocannabinoid generation and reinforce the view of GPR55 and LPI being genuine actors of the endocannabinoid system.

Phosphoinositide 3-kinases in platelets, thrombosis and therapeutics.

Our knowledge on the expression, regulation and roles of the different phosphoinositide 3-kinases (PI3Ks) in platelet signaling and functions has greatly expanded these last twenty years. Much progress has been made in understanding the roles and regulations of class I PI3Ks which produce the lipid second messenger phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3). Selective pharmacological inhibitors and genetic approaches have allowed researchers to generate an impressive amount of data on the role of class I PI3Kα, ß, δ and γ in platelet activation and in thrombosis. Furthermore, platelets do also express two class II PI3Ks (PI3KC2α and PI3KC2ß), thought to generate PtdIns(3,4)P2 and PtdIns3P, and the sole class III PI3K (Vps34), known to synthesize PtdIns3P. Recent studies have started to reveal the importance of PI3KC2α and Vps34 in megakaryocytes and platelets, opening new perspective in our comprehension of platelet biology and thrombosis. In this review, we will summarize previous and recent advances on platelet PI3Ks isoforms. The implication of these kinases and their lipid products in fundamental platelet biological processes and thrombosis will be discussed. Finally, the relevance of developing potential antithrombotic strategies by targeting PI3Ks will be examined.

A dual role for the class III PI3K, Vps34, in platelet production and thrombus growth.

To uncover the role of Vps34, the sole class III phosphoinositide 3-kinase (PI3K), in megakaryocytes (MKs) and platelets, we created a mouse model with Vps34 deletion in the MK/platelet lineage (Pf4-Cre/Vps34lox/lox). Deletion of Vps34 in MKs led to the loss of its regulator protein, Vps15, and was associated with microthrombocytopenia and platelet granule abnormalities. Although Vps34 deficiency did not affect MK polyploidisation or proplatelet formation, it dampened MK granule biogenesis and directional migration toward an SDF1α gradient, leading to ectopic platelet release within the bone marrow. In MKs, the level of phosphatidylinositol 3-monophosphate (PI3P) was significantly reduced by Vps34 deletion, resulting in endocytic/trafficking defects. In platelets, the basal level of PI3P was only slightly affected by Vps34 loss, whereas the stimulation-dependent pool of PI3P was significantly decreased. Accordingly, a significant increase in the specific activity of Vps34 lipid kinase was observed after acute platelet stimulation. Similar to Vps34-deficient platelets, ex vivo treatment of wild-type mouse or human platelets with the Vps34-specific inhibitors, SAR405 and VPS34-IN1, induced abnormal secretion and affected thrombus growth at arterial shear rate, indicating a role for Vps34 kinase activity in platelet activation, independent from its role in MKs. In vivo, Vps34 deficiency had no impact on tail bleeding time, but significantly reduced platelet prothrombotic capacity after carotid injury. This study uncovers a dual role for Vps34 as a regulator of platelet production by MKs and as an unexpected regulator of platelet activation and arterial thrombus formation dynamics.

Profiling of phosphoinositide molecular species in human and mouse platelets identifies new species increasing following stimulation.

Phosphoinositides are bioactive lipids essential in the regulation of cell signaling as well as cytoskeleton and membrane dynamics. Their metabolism is highly active in blood platelets where they play a critical role during activation, at least through two well identified pathways involving phospholipase C and phosphoinositide 3-kinases (PI3K). Here, using a sensitive high-performance liquid chromatography-mass spectrometry method recently developed, we monitored for the first time the profiling of phosphatidylinositol (PI), PIP, PIP2 and PIP3 molecular species (fatty-acyl profiles) in human and mouse platelets during the course of stimulation by thrombin and collagen-related peptide. Furthermore, using class IA PI3K p110α or p110ß deficient mouse platelets and a pharmacological inhibitor, we show the crucial role of p110ß and the more subtle role of p110α in the production of PIP3 molecular species following stimulation. This comprehensive platelet phosphoinositides profiling provides important resources for future studies and reveals new information on phosphoinositides biology, similarities and differences in mouse and human platelets and unexpected dramatic increase in low-abundance molecular species of PIP2 during stimulation, opening new perspectives in phosphoinositide signaling in platelets.

Functional Characterization and Rescue of a Deep Intronic Mutation in OCRL Gene Responsible for Lowe Syndrome.

Dent-2 disease and Lowe syndrome are two pathologies caused by mutations in inositol polyphosphate 5-phosphatase OCRL gene. Both conditions share proximal tubulopathy evolving to chronic kidney failure. Lowe syndrome is in addition defined by a bilateral congenital cataract, intellectual disability, and hypotonia. The pathology evolves in two decades to a severe condition with renal complications and a fatal issue. We describe here a proof of principle for a targeted gene therapy on a mutation of the OCRL gene that is associated with Lowe syndrome. The affected patient bears a deep intronic mutation inducing a pseudo-exon inclusion in the mRNA, leading to a OCRL-1 protein loss. An exon-skipping strategy was designed to correct the effect of the mutation in cultured cells. We show that a recombinant U7-modified small RNA efficiently triggered the restoration of normal OCRL expression at mRNA and protein levels in patient's fibroblasts. Moreover, the PI(4,5)P2 accumulation and cellular alterations that are hallmark of OCRL-1 dysfunction were also rescued. Altogether, we provide evidence that the restoration of OCRL-1 protein, even at a reduced level, through RNA-based therapy represents a potential therapeutic approach for patients with OCRL splice mutations.

TOM1 is a PI5P effector involved in the regulation of endosomal maturation.

Phosphoinositides represent a major class of lipids specifically involved in the organization of signaling cascades, maintenance of the identity of organelles and regulation of multiple intracellular trafficking steps. We previously reported that phosphatidylinositol 5-monophosphate (PI5P), produced by the Shigella flexneri phosphatase IpgD, is implicated in the endosomal sorting of the epidermal growth factor receptor (EGFR). Here, we show that the adaptor protein TOM1 is a new direct binding partner of PI5P. We identify the domain of TOM1 involved in this interaction and characterize the binding motif. Finally, we demonstrate that the recruitment of TOM1 by PI5P on signaling endosomes is responsible for the delay in EGFR degradation and fluid-phase bulk endocytosis. Taken together, our data strongly suggest that PI5P enrichment in signaling endosomes prevents endosomal maturation through the recruitment of TOM1, and point to a new function of PI5P in regulating discrete maturation steps in the endosomal system.

Essential role of class II PI3K-C2α in platelet membrane morphology.

The physiologic roles of the class II phosphoinositide 3-kinases (PI3Ks) and their contributions to phosphatidylinositol 3-monophosphate (PI3P) and PI(3,4)P2 production remain elusive. Here we report that mice heterozygous for a constitutively kinase-dead PI3K-C2α display aberrant platelet morphology with an elevated number of barbell-shaped proplatelets, a recently discovered intermediate stage in the final process of platelet production. Platelets with heterozygous PI3K-C2α inactivation have critical defects in α-granules and membrane structure that are associated with modifications in megakaryocytes. These platelets are more rigid and unable to form filopodia after stimulation. Heterozygous PI3K-C2α inactivation in platelets led to a significant reduction in the basal pool of PI3P and a mislocalization of several membrane skeleton proteins known to control the interactions between the plasma membrane and cytoskeleton. These alterations had repercussions on the performance of platelet responses with delay in the time of arterial occlusion in an in vivo model of thrombosis and defect in thrombus formation in an ex vivo blood flow system. These data uncover a key role for PI3K-C2α activity in the generation of a basal housekeeping PI3P pool and in the control of membrane remodeling, critical for megakaryocytopoiesis and normal platelet production and function.

Myotubularin and PtdIns3P remodel the sarcoplasmic reticulum in muscle in vivo.

The sarcoplasmic reticulum (SR) is a specialized form of endoplasmic reticulum (ER) in skeletal muscle and is essential for calcium homeostasis. The mechanisms involved in SR remodeling and maintenance of SR subdomains are elusive. In this study, we identified myotubularin (MTM1), a phosphoinositide phosphatase mutated in X-linked centronuclear myopathy (XLCNM, or myotubular myopathy), as a key regulator of phosphatidylinositol 3-monophosphate (PtdIns3P) levels at the SR. MTM1 is predominantly located at the SR cisternae of the muscle triads, and Mtm1-deficient mouse muscles and myoblasts from XLCNM patients exhibit abnormal SR/ER networks. In vivo modulation of MTM1 enzymatic activity in skeletal muscle using ectopic expression of wild-type or a dead-phosphatase MTM1 protein leads to differential SR remodeling. Active MTM1 is associated with flat membrane stacks, whereas dead-phosphatase MTM1 mutant promotes highly curved cubic membranes originating from the SR and enriched in PtdIns3P. Overexpression of a tandem FYVE domain with high affinity for PtdIns3P alters the shape of the SR cisternae at the triad. Our findings, supported by the parallel analysis of the Mtm1-null mouse and an in vivo study, reveal a direct function of MTM1 enzymatic activity in SR remodeling and a key role for PtdIns3P in promoting SR membrane curvature in skeletal muscle. We propose that alteration in SR remodeling is a primary cause of X-linked centronuclear myopathy. The tight regulation of PtdIns3P on specific membrane subdomains may be a general mechanism to control membrane curvature.

Phosphatase-dead myotubularin ameliorates X-linked centronuclear myopathy phenotypes in mice.

Myotubularin MTM1 is a phosphoinositide (PPIn) 3-phosphatase mutated in X-linked centronuclear myopathy (XLCNM; myotubular myopathy). We investigated the involvement of MTM1 enzymatic activity on XLCNM phenotypes. Exogenous expression of human MTM1 in yeast resulted in vacuolar enlargement, as a consequence of its phosphatase activity. Expression of mutants from patients with different clinical progression and determination of PtdIns3P and PtdIns5P cellular levels confirmed the link between vacuolar morphology and MTM1 phosphatase activity, and showed that some disease mutants retain phosphatase activity. Viral gene transfer of phosphatase-dead myotubularin mutants (MTM1(C375S) and MTM1(S376N)) significantly improved most histological signs of XLCNM displayed by a Mtm1-null mouse, at similar levels as wild-type MTM1. Moreover, the MTM1(C375S) mutant improved muscle performance and restored the localization of nuclei, triad alignment, and the desmin intermediate filament network, while it did not normalize PtdIns3P levels, supporting phosphatase-independent roles of MTM1 in maintaining normal muscle performance and organelle positioning in skeletal muscle. Among the different XLCNM signs investigated, we identified only triad shape and fiber size distribution as being partially dependent on MTM1 phosphatase activity. In conclusion, this work uncovers MTM1 roles in the structural organization of muscle fibers that are independent of its enzymatic activity. This underlines that removal of enzymes should be used with care to conclude on the physiological importance of their activity.

cdc-like/dual-specificity tyrosine phosphorylation-regulated kinases inhibitor leucettine L41 induces mTOR-dependent autophagy: implication for Alzheimer's disease.

Leucettines, a family of pharmacological inhibitors of dual-specificity tyrosine phosphorylation regulated kinases and cdc-like kinases (CLKs), are currently under investigation for their potential therapeutic application to Down syndrome and Alzheimer's disease. We here report that leucettine L41 triggers bona fide autophagy in osteosarcoma U-2 OS cells and immortalized mouse hippocampal HT22 cells, characterized by microtubule-associated protein light chain 3 membrane translocation and foci formation. Leucettine L41-triggered autophagy requires the Unc-51-like kinase and is sensitive to the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and 3-methyladenine, suggesting that it acts through the mammalian target of rapamycin (mTOR)/PI3K-dependent pathway. Leucettine L41 does not act by modifying the autophagic flux of vesicles. Leucettine L41-induced autophagy correlates best with inhibition of CLKs. Leucettine L41 modestly inhibited phosphatidylinositol-3-phosphate 5-kinase, FYVE domain-containing activity as tested both in vitro and in vivo, which may also contribute to autophagy induction. Altogether these results demonstrate that leucettines can activate the autophagic mTOR/PI3K pathway, a characteristic that may turn advantageous in the context of Alzheimer's disease treatment.

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.