Targeted metabolomic analyses of cellular models of pelizaeus-merzbacher disease reveal plasmalogen and myo-inositol solute carrier dysfunction.
ABSTRACT: BACKGROUND: Leukodystrophies are devastating diseases characterized by dys- and hypo-myelination. While there are a number of histological and imaging studies of these disorders, there are limited biochemical data available. We undertook targeted lipidomic analyses of Pelizaeus-Merzbacher disease (PMD) fibroblasts, PMD lymphocytes, and 158JP oligodendrocytes, a murine model of PMD, to define the lipid changes in these cell models. Further targeted metabolomics analyses were conducted to obtain a preliminary evaluation of the metabolic consequences of lipid changes and gene mutations in these cell models. RESULTS: In both PMD fibroblasts and lymphocytes, and 158JP oligodendrocytes, ethanolamine plasmalogens were significantly decreased. Labeling studies with 158JP oligodendrocytes further demonstrated a decreased rate of lipid remodeling at sn-2. Targeted metabolomics analyses of these cells revealed dramatic increases in cellular levels of myo-inositol. Further uptake studies demonstrated increased rates of myo-inositol uptake by PMD lymphocytes. CONCLUSIONS: Our data demonstrating PlsEtn decrements, support previous studies indicating leukodystrophy cells possess significant peroxisomal deficits. Our data for the first time also demonstrate that decrements in peroxisomal function coupled with the PLP1 gene defects of PMD, result in changes in the function of membrane myo-inositol solute carriers resulting in dramatic increases in cellular myo-inositol levels.
Project description:BACKGROUND: Childhood peroxisomal disorders and leukodystrophies are devastating diseases characterized by dysfunctional lipid metabolism. Plasmalogens (ether glycerophosphoethanolamine lipids) are decreased in these genetic disorders. The biosynthesis of plasmalogens is initiated in peroxisomes but completed in the endoplasmic reticulum. We therefore undertook a study to evaluate the ability of a 3-substituted, 1-alkyl, 2-acyl glyceryl ether lipid (PPI-1011) to replace plasmalogens in rhizomelic chrondrodysplasia punctata type 1 (RCDP1) and rhizomelic chrondrodysplasia punctata type 2 (RCDP2) lymphocytes which possess peroxisomal mutations culminating in deficient plasmalogen synthesis. We also examined plasmalogen synthesis in Pelizaeus-Merzbacher disease (PMD) lymphocytes which possess a proteolipid protein-1 (PLP1) missense mutation that results in abnormal PLP1 folding and it's accumulation in the endoplasmic reticulum (ER), the cellular site of the last steps in plasmalogen synthesis. In vivo incorporation of plasmalogen precursor into tissue plasmalogens was also evaluated in the Pex7 mouse model of plasmalogen deficiency. RESULTS: In both RCDP1 and RCDP2 lymphocytes, PPI-1011 repleted the target ethanolamine plasmalogen (PlsEtn16:0/22:6) in a concentration dependent manner. In addition, deacylation/reacylation reactions resulted in repletion of PlsEtn 16:0/20:4 in both RCDP1 and RCDP2 lymphocytes, repletion of PlsEtn 16:0/18:1 and PlsEtn 16:0/18:2 in RCDP2 lymphocytes, and partial repletion of PlsEtn 16:0/18:1 and PlsEtn 16:0/18:2 in RCDP1 lymphocytes. In the Pex7 mouse, oral dosing of labeled PPI-1011 demonstrated repletion of tissue levels of the target plasmalogen PlsEtn 16:0/22:6 with phospholipid remodeling also resulting in significant repletion of PlsEtn 16:0/20:4 and PlsEtn 16:0/18:1. Metabolic conversion of PPI-1011 to the target plasmalogen was most active in the liver. CONCLUSIONS: Our data demonstrate that PPI-1011 is activated (removal of 3-substitution) and converted to PlsEtn in vitro in both RCDP1 and RCDP2 lymphocytes and in vivo in the Pex7 mouse model of RCPD1 effectively bypassing the peroxisomal dysfunction present in these disorders. While PPI-1011 was shown to replete PlsEtns 16:0/x, ether lipid precursors of PlsEtn 18:0/x and PlsEtn 18:1/x may also be needed to achieve optimal clinical benefits of plasmalogen replacement in these complex patient populations. In contrast, only limited plasmalogen replacement was observed in PMD lymphocytes suggesting that the effects of protein misfolding and accumulation in the ER negatively affect processing of plasmalogen precursors in this cellular compartment.
Project description:Entamoeba histolytica is believed to be devoid of peroxisomes, like most anaerobic protists. In this work, we provided the first evidence that peroxisomes are present in E. histolytica, although only seven proteins responsible for peroxisome biogenesis (peroxins) were identified (Pex1, Pex6, Pex5, Pex11, Pex14, Pex16, and Pex19). Targeting matrix proteins to peroxisomes is reduced to the PTS1-dependent pathway mediated via the soluble Pex5 receptor, while the PTS2 receptor Pex7 is absent. Immunofluorescence microscopy showed that peroxisomal markers (Pex5, Pex14, Pex16, Pex19) are present in vesicles distinct from mitosomes, the endoplasmic reticulum, and the endosome/phagosome system, except Pex11, which has dual localization in peroxisomes and mitosomes. Immunoelectron microscopy revealed that Pex14 localized to vesicles of approximately 90-100 nm in diameter. Proteomic analyses of affinity-purified peroxisomes and in silico PTS1 predictions provided datasets of 655 and 56 peroxisomal candidates, respectively; however, only six proteins were shared by both datasets, including myo-inositol dehydrogenase (myo-IDH). Peroxisomal NAD-dependent myo-IDH appeared to be a dimeric enzyme with high affinity to myo-inositol (Km 0.044 mM) and can utilize also scyllo-inositol, D-glucose and D-xylose as substrates. Phylogenetic analyses revealed that orthologs of myo-IDH with PTS1 are present in E. dispar, E. nutalli and E. moshkovskii but not in E. invadens, and form a monophyletic clade of mostly peroxisomal orthologs with free-living Mastigamoeba balamuthi and Pelomyxa schiedti. The presence of peroxisomes in E. histolytica and other archamoebae breaks the paradigm of peroxisome absence in anaerobes and provides a new potential target for the development of antiparasitic drugs.
Project description:Hepatocellular carcinoma (HCC) is one of the commonest causes of death from cancer. A plethora of metabolomic investigations of HCC have yielded molecules in biofluids that are both up- and down-regulated but no real consensus has emerged regarding exploitable biomarkers for early detection of HCC. We report here a different approach, a combined transcriptomics and metabolomics study of energy metabolism in HCC. A panel of 31 pairs of HCC tumors and corresponding nontumor liver tissues from the same patients was investigated by gas chromatography-mass spectrometry (GCMS)-based metabolomics. HCC was characterized by ?2-fold depletion of glucose, glycerol 3- and 2-phosphate, malate, alanine, myo-inositol, and linoleic acid. Data are consistent with a metabolic remodeling involving a 4-fold increase in glycolysis over mitochondrial oxidative phosphorylation. A second panel of 59 HCC that had been typed by transcriptomics and classified in G1 to G6 subgroups was also subjected to GCMS tissue metabolomics. No differences in glucose, lactate, alanine, glycerol 3-phosphate, malate, myo-inositol, or stearic acid tissue concentrations were found, suggesting that the Wnt/?-catenin pathway activated by CTNNB1 mutation in subgroups G5 and G6 did not exhibit specific metabolic remodeling. However, subgroup G1 had markedly reduced tissue concentrations of 1-stearoylglycerol, 1-palmitoylglycerol, and palmitic acid, suggesting that the high serum ?-fetoprotein phenotype of G1, associated with the known overexpression of lipid catabolic enzymes, could be detected through metabolomics as increased lipid catabolism.Tissue metabolomics yielded precise biochemical information regarding HCC tumor metabolic remodeling from mitochondrial oxidation to aerobic glycolysis and the impact of molecular subtypes on this process.
Project description:E. histolytica is believed to be devoid of peroxisomes, like most anaerobic protists. In this work, we provided the first evidence that peroxisomes are present in E. histolytica, although the set of proteins (peroxins) responsible for peroxisome biogenesis was reduced to only seven members (Pex1, Pex6, Pex5, Pex11, Pex14, Pex16, and Pex19). Targeting matrix proteins to peroxisomes is reduced to the PTS1-dependent pathway mediated via the soluble Pex5 receptor, while the PTS2 receptor Pex7 is absent. Immunofluorescence microscopy showed that peroxisomal markers (Pex5, Pex14, Pex16, Pex19) are present in vesicles distinct from mitosomes, the endoplasmic reticulum and the endosome/phagosome system, except Pex11, which has dual localization in peroxisomes and mitosomes. Immunoelectron microscopy revealed that Pex14 localized to vesicles of approximately 90-100 nm in diameter. Proteomic analyses of affinity-purified peroxisomes and in silico PTS1 predictions provided datasets of 655 and 56 peroxisomal candidates, respectively; however, only six proteins were shared by both datasets, including myo-inositol dehydrogenase (myo-IDH). Peroxisomal NAD-dependent myo-IDH appeared to be a dimeric enzyme with high affinity to myo-inositol (Km 0,044 mM) and can utilize also scyllo-inositol, D-glucose and D-xylose as substrates. Phylogenetic analyses revealed that orthologs of myo-IDH with PTS1 are present in E. dispar, E. nutalli and E. moshkovskii but not in E. invadens, and form a monophyletic clade of mostly peroxisomal orthologs with free-living M. balamuthi and P. schiedti. The presence of peroxisomes in E. histolytica and other archamoebae breaks the paradigm of peroxisome absence in anaerobes and provides a new potential target for the development of antiparasitic drugs.
Project description:Radioactive myo-inositol was injected intraperitoneally into nephrectomized rats. The radioactive material present in liver, spleen, brain, heart, diaphragm, seminal vesicle, coagulating gland, prostate, epididymis, vas deferens and testis was shown to consist exclusively of myo-inositol and its derivatives, as shown by paper chromatography of hydrolysates and trichloroacetic acid extracts of these tissues. Radioactive myo-inositol was accumulated rapidly within 1 h by the thyroid, coagulating gland and seminal vesicle. Other tissues, such as the pituitary, prostate gland, liver and spleen, concentrated myo-inositol less actively. The muscle tissues studied (diaphragm and heart) concentrated little inositol, whereas brain, testis, and epididymal fat-pad did not concentrate it at all. The lipid fraction of liver contained most of the radio-labelled myo-inositol. In the other organs most of the radioactivity was found in the aqueous trichloroacetic acid extract, largely as free myo-inositol.
Project description:myo-Inositol analysis of detergent-solubilized immunoaffinity-purified rat liver 5'-nucleotidase showed the presence of 1 mol of myo-inositol/mol of enzyme monomer. This provides unequivocal evidence that the ectoenzyme 5'-nucleotidase is attached to liver membranes by a glycosyl-phosphatidylinositol lipid anchor.
Project description:The aim of the study was to characterize the soluble metabolomics profile of defatted colostrum of sows at different parity number (PA) and to correlate the metabolomics profile with the Brix percentage estimate of colostrum immunoglobulin G (IgG) and sow productive traits. A total of 96 Meidam (crossbreed Large White × Meishan) sows of PA from 1-4 (PA1: 28; PA2:26; PA3:12; PA4:26) were included, and their productive traits were recorded at 10 days post-farrowing. Colostrum IgG was quantified using a Brix refractometer, and metabolomics profile was assessed using 1H-NMR spectroscopy. Sows' PA slightly influenced the metabolomics profile of colostrum. lactose and glycine were higher in PA1 compared with PA4 (p 0.05) and N-acetylglucosamine (GlcNAc) tended to be higher in PA2 than PA3 and PA4 (p < 0.10). The Brix percentage of IgG was negatively associated with lactose and positively with creatine, myo-inositol, and O-phosphocholine (p < 0.05). Taurine was positively related to litter weight at birth. GlcNAc and myo-inositol were linked to piglet mortality at day 10 with a negative and positive trend, respectively. In conclusion, colostrum of gilts and multiparous sows had a similar metabolomics profile. Specific metabolites contributed to explanation of the variability in colostrum Brix percentage estimate of IgG concentration and the sows' productive performance.
Project description:Pelizaeus-Merzbacher disease (PMD) is a form of X-linked leukodystrophy caused by mutations in the proteolipid protein 1 (PLP1) gene. Although PLP1 proteins with missense mutations have been shown to accumulate in the rough endoplasmic reticulum (ER) in disease model animals and cell lines transfected with mutant PLP1 genes, the exact pathogenetic mechanism of PMD has not previously been clarified. In this study, we established induced pluripotent stem cells (iPSCs) from two PMD patients carrying missense mutation and differentiated them into oligodendrocytes in vitro. In the PMD iPSC-derived oligodendrocytes, mislocalization of mutant PLP1 proteins to the ER and an association between increased susceptibility to ER stress and increased numbers of apoptotic oligodendrocytes were observed. Moreover, electron microscopic analysis demonstrated drastically reduced myelin formation accompanied by abnormal ER morphology. Thus, this study demonstrates the involvement of ER stress in pathogenic dysmyelination in the oligodendrocytes of PMD patients with the PLP1 missense mutation.
Project description:Type II pneumonocytes isolated from adult rat lung were incubated in a serum-free medium containing [14C]glycerol and the incorporation of 14C into glycerophospholipids was measured. After 24 h, more than 80% of the 14C incorporated into total lipids or into phosphatidylcholine and approx. 90% of the 14C incorporated into phosphatidylglycerol after 24 h was recovered in the glycerophosphoester moieties of these molecules. Supplementation of the incubation medium with foetal-bovine serum (10%, v/v) did not alter the incorporation of [14C]glycerol by type II pneumonocytes after 24 h into either a total lipid extract or phosphatidylcholine. In the presence of foetal-bovine serum, however, the incorporation of 14C into phosphatidylglycerol was decreased and the incorporation of 14C into phosphatidylinositol was increased. In the absence of foetal-bovine serum, the incorporation of 14C into phosphatidylglycerol was decreased progressively as the concentration of myo-inositol in the incubation medium was increased. The range of concentration (0.04-0.50 mM) over which myo-inositol had the greatest influence on [14C]glycerol incorporation into phosphatidylglycerol by type II pneumonocytes in vitro encompassed the concentration range measured in foetal-rat serum late in gestation. At 4 days before birth, the concentration of myo-inositol in foetal-rat serum was 0.36 mM and decreased to 0.23 mM 1 day before birth. The concentration of myo-inositol in adult rat serum increased from 0.03 mM to 0.06 mM during pregnancy. Isolated rat type II pneumonocytes were found to take up myo-inositol by a saturable process. A half-maximal rate of myo-inositol uptake occurred at a concentration of myo-inositol of 0.29 mM. The results of this investigation are consistent with the hypothesis that late in gestation there is a decreasing availability of myo-inositol to the foetal lungs and that this favours the biosynthesis of phosphatidylglycerol for surfactant at the expense of phosphatidylinositol biosynthesis.
Project description:We report next generation sequencing RNA-seq data of human gut commensal Bacteroides thetaiotaomicron strains deficient in inositol lipid synthesis, including dBT_1522 (phosphoinositol dihydroceramide synthase knockout) and its wild-type background strain, and iSPTdBT_1526 (myo-inositol-phosphate synthase) knockout with its background strain ("iSPT," inducible serine palmitoyltransferase).