Genetic Interaction between MTMR2 and FIG4 Phospholipid Phosphatases Involved in Charcot-Marie-Tooth Neuropathies
Ilaria VaccariGiorgia DinaHélène TronchèreEmily KaufmanGaëtan ChicanneFederica CerriLawrence WrabetzBernard PayrastreAngelo QuattriniLois S. WeismanMiriam H. MeislerAlessandra Bolino
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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)P2, 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)P2 and MTMR2 catalyzes its dephosphorylation, these two phosphatases might be expected to have opposite effects in the control of PtdIns(3,5)P2 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)P2 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.Abstract— Gangliosides were isolated from myelin prepared from mouse brains of different ages (23 to 490 days). Quantitative estimation of lipid‐bound sialic acid levels indicated a gradual increase from 560 μg/g of myelin at 23 days to about 1200 μg/g of myelin at older ages. The major ganglioside in all myelin preparations was the monosialoganglioside G 4 (G M1 ). However, considerable amounts of di‐ and trisialo species also were found in myelin from young animals. In contrast to human myelin in which the monosialoganglioside, sialosylgalactosylceramide (G 7 ) was highly enriched (L edeen et al. , 1973), a much smaller enrichment of this ganglioside was noticed in mouse brain myelin. Ganglioside G 7 was not detectable in myelin until the animals were 35 days old, and showed a slight increase with increasing age after that. The results strongly indicated that the concentration of G 7 in myelin is species specific and age dependent. The study also demonstrated that the ganglioside accretion in developing mouse brain myelin was attributable to the enrichment of monosialogangliosides G 4 (G M1 ), G 5 (G M2 ) and G 7 at the expense of polysialogangliosides.
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Abstract: At intervals ranging from 1 to 10 min after injection of 32 Pi into rat brain, myelin was prepared and separated into three subfractions: heavy, medium, and light. The radioactivity of total phospholipids and polyphospho‐inositides (PPI) was then determined. There was rapid incorporation of 32 Pi into PPI, which contained 50–70% of the radioactivity among total brain lipids and more than 70% among myelin lipids. The myelin fraction had incorporated 32 Pi into total recovered PPI in the order of medium > heavy > light fraction: however, the order of relative specific radioactivities was heavy > light > medium. Labeling of the PPI precursors, phosphatidic acid (PA) and phos‐phatidylinositol (PI), was considerably lower in the purified myelin than in total brain. The di‐ (DPI) and triphosphoinositides (TPI) in heavy myelin exchanged 32 Pi at rates 2 to 3 times faster than those in medium and light myelin. DPI of all subfractions of myelin exchanged much faster than TPI. The results show that the most active phosphate turnover of myelin PPI occurs in the heavy myelin fraction (probably largely consisting of myelin appurtenant regions). However, medium and light myelin (most probably representing the closely packed layers of myelin sheaths) also showed rapid turnover of PPI.
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L1210 murine leukemic cells were serially passaged in BDF1 mice and were treated in vivo with methotrexate (MTX) and 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP) alone or in combination. The time course of emergence of resistance of the treated cell lines was studied both in vivo and in vitro. When used as a single drug, in vivo resistance to MTX developed gradually and was considerable at eight passages and complete by 11 passages. Complete in vivo resistance to DDMP used alone occurred by the fifth passage. Complete in vivo resistance to the drugs in combination was seen by the eighth passage. In cells demonstrating complete DDMP resistance, as determined in vivo, there was no evidence of cross-resistance to MTX measured either in vivo or in vitro, while MTX resistance was associated with incomplete cross-resistance to DDMP. The greatest degree of resistance, as determined in vitro, occurred in the cell line treated with the drug combination. In vitro tests of drug resistance correlated well with in vivo survival data. An important observation was that major in vivo drug resistance was accompanied by only a small measurable effect using standard in vitro screening techniques. The implications of this finding are discussed.
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Abstract In the following review, we address difficulties that have arisen when attempting to convert the myelin multilayers into vesicles. The emphasis is on CNS myelin of adult mammals although both central nervous system (CNS) and peripheral nervous system (PNS) myelin are considered. The ability to prepare vesicle of myelin membrane has yet not been feasible. We hope to clarify some aspect of this problem and offer some possible approaches. Special attention is paid to myelin swelling phenomena because these indicate ways in which the myelin multilayer can break down. Images of isolated myelin are reviewed with special attention to the ways in which the multilayer actually breaks down. Attempts at reproducing a procedure for vesiculating myelin are summarized, and a critique is given to account for the inability to reproduce the published results. Finally, novel approaches for vesiculating myelin are proposed, which are based on well‐characterized swelling phenomena. © 1995 Wiley‐Liss, Inc.
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Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (CζAGΨGR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates.
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In vitro determination of metabolic stability is routinely used to assess the overall metabolic liability of compounds and for prioritization for in vivo studies. If in vitro metabolic stability data could be used to reliably predict in vivo clearance (CL), it would add significant value in the selection of compounds for in vivo pharmacokinetic and pharmacology studies. We have evaluated the utility of our in vitro metabolic stability screening assay to estimate in vivo CL in the mouse. The in vitro mouse clearances (CLin vitro) of 146 structurally diverse compounds with metabolic stabilities > 30 %, were compared to mouse in vivo CL data. Approximately 45 % of the compounds showed agreement between in vivo CL and predicted CLin vitro within a 2-fold error criteria. The correlation appeared worse when correction for the extent of incorporation of plasma protein binding or both plasma and S9 bindings (i.e. ~14 % and~ 28 % agreement, respectively). Classification of the compounds into three groups based on in vivo CL (<30 mL/min/kg, 30-70 mL/min/kg, and >70 mL/min/kg) did not show any improvement between in vivo CL and predicted CLin vitro. The percentage of compounds falling within the 2-fold error criteria for low CL, moderate CL and high CL groups were 54, 31 and 24 %, respectively. In conclusion, our analysis suggests that in vitro metabolic stability data, as routinely obtained in early ADME screening protocols, does not demonstrate a strong correlation with or predictivity for, absolute in vivo CL in the mouse.
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