The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Stephane Savary - One of the best experts on this subject based on the ideXlab platform.
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crispr cas9 mediated knockout of ABCD1 and abcd2 genes in bv 2 cells novel microglial models for x linked adrenoleukodystrophy
Biochimica et Biophysica Acta, 2019Co-Authors: Quentin Raas, Catherine Gondcaille, Doriane Trompier, Franck Ménétrier, Yannick Hamon, Gérard Lizard, Valerio Leoni, Claudio Caccia, Stephane SavaryAbstract:X-linked adrenoleukodystrophy (X-ALD), the most frequent peroxisomal disorder, is associated with mutation in the ABCD1 gene which encodes a peroxisomal ATP-binding cassette transporter for very long-chain fatty acids (VLCFA). The biochemical hallmark of the disease is the accumulation of VLCFA. Peroxisomal defect in microglia being now considered a priming event in the pathology, we have therefore generated murine microglial cells mutated in the ABCD1 gene and its closest homolog, the Abcd2 gene. Using CRISPR/Cas9 gene editing strategy, we obtained 3 cell clones with a single or double deficiency. As expected, only the combined absence of ABCD1 and ABCD2 proteins resulted in the accumulation of VLCFA. Ultrastructural analysis by electron microscopy revealed in the double mutant cells the presence of lipid inclusions similar to those observed in brain macrophages of patients. These observations are likely related to the increased level of cholesterol and the accumulation of neutral lipids that we noticed in mutant cells. A preliminary characterization of the impact of peroxisomal defects on the expression of key microglial genes such as Trem2 suggests profound changes in microglial functions related to inflammation and phagocytosis. The expression levels of presumed modifier genes have also been found modified in mutant cells, making these novel cell lines relevant for use as in vitro models to better understand the physiopathogenesis of X-ALD and to discover new therapeutic targets.
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CRISPR/Cas9-mediated knockout of ABCD1 and Abcd2 genes in BV-2 cells: novel microglial models for X-linked Adrenoleukodystrophy
Biochimica Et Biophysica Acta-Molecular and Cell Biology of Lipids, 2019Co-Authors: Quentin Raas, Catherine Gondcaille, Doriane Trompier, Franck Ménétrier, Yannick Hamon, Gérard Lizard, Valerio Leoni, Claudio Caccia, Stephane SavaryAbstract:X-linked adrenoleukodystrophy (X-ALD), the most frequent peroxisomal disorder, is associated with mutation in the ABCD1 gene which encodes a peroxisomal ATP-binding cassette transporter for very long-chain fatty acids (VLCFA). The biochemical hallmark of the disease is the accumulation of VLCFA. Peroxisomal defect in microglia being now considered a priming event in the pathology, we have therefore generated murine microglial cells mutated in the ABCD1 gene and its closest homolog, the Abcd2 gene. Using CRISPR/Cas9 gene editing strategy, we obtained 3 cell clones with a single or double deficiency. As expected, only the combined absence of ABCD1 and ABCD2 proteins resulted in the accumulation of VLCFA. Ultrastructural analysis by electron microscopy revealed in the double mutant cells the presence of lipid inclusions similar to those observed in brain macrophages of patients. These observations are likely related to the increased level of cholesterol and the accumulation of neutral lipids that we noticed in mutant cells. A preliminary characterization of the impact of peroxisomal defects on the expression of key microglial genes such as Trem2 suggests profound changes in microglial functions related to inflammation and phagocytosis. The expression levels of presumed modifier genes have also been found modified in mutant cells, making these novel cell lines relevant for use as in vitro models to better understand the physiopathogenesis of X-ALD and to discover new therapeutic targets.
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Predictive Structure and Topology of Peroxisomal ATP-Binding Cassette (ABC) Transporters
MDPI AG, 2017Co-Authors: Pierre Andreoletti, Catherine Gondcaille, Doriane Trompier, Mustapha Cherkaoui-malki, Quentin Raas, Stephane SavaryAbstract:The peroxisomal ATP-binding Cassette (ABC) transporters, which are called ABCD1, ABCD2 and ABCD3, are transmembrane proteins involved in the transport of various lipids that allow their degradation inside the organelle. Defective ABCD1 leads to the accumulation of very long-chain fatty acids and is associated with a complex and severe neurodegenerative disorder called X-linked adrenoleukodystrophy (X-ALD). Although the nucleotide-binding domain is highly conserved and characterized within the ABC transporters family, solid data are missing for the transmembrane domain (TMD) of ABCD proteins. The lack of a clear consensus on the secondary and tertiary structure of the TMDs weakens any structure-function hypothesis based on the very diverse ABCD1 mutations found in X-ALD patients. Therefore, we first reinvestigated thoroughly the structure-function data available and performed refined alignments of ABCD protein sequences. Based on the 2.85 Å resolution crystal structure of the mitochondrial ABC transporter ABCB10, here we propose a structural model of peroxisomal ABCD proteins that specifies the position of the transmembrane and coupling helices, and highlight functional motifs and putative important amino acid residues
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regulation of the adrenoleukodystrophy related gene abcd2 focus on oxysterols and lxr antagonists
Biochemical and Biophysical Research Communications, 2014Co-Authors: Doriane Trompier, Catherine Gondcaille, Gérard Lizard, Stephane SavaryAbstract:Abstract The regulation of the ABCD2 gene is recognized as a possible therapeutic target for X-linked adrenoleukodystrophy, a rare neurodegenerative disease caused by mutations in the ABCD1 gene. Up-regulation of ABCD2 expression has indeed been demonstrated to compensate for ABCD1 deficiency, restoring peroxisomal β-oxidation of very-long-chain fatty acids. Besides the known inducers of the ABCD2 gene (phenylbutyrate and histone deacetylase inhibitors, fibrates, dehydroepiandrosterone, thyroid hormone and thyromimetics), this review will focus on LXR antagonists and 22S-hydroxycholesterol, recently described as inducers of ABCD2 expression. Several LXR antagonists have been identified and their possible indication for neurodegenerative disorders will be discussed.
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Peroxisomal and mitochondrial status of two murine oligodendrocytic cell lines (158N, 158JP): potential models for the study of peroxisomal disorders associated with dysmyelination processes.
Journal of Neurochemistry, 2009Co-Authors: Mauhamad Baarine, Doriane Trompier, Pierre Andreoletti, Kevin Ragot, Hammam El Hajj, M Said Ghandour, Franck Ménétrier, Mustapha Cherkaoui-malki, Emmanuelle Genin, Stephane SavaryAbstract:In some neurodegenerative disorders (leukodystrophies) characterized by myelin alterations, the defect of peroxisomal functions on myelin-producing cells (oligodendrocytes) are poorly understood. The development of in vitro models is fundamental to understanding the physiopathogenesis of these diseases. We characterized two immortalized murine oligodendrocyte cell lines: a normal (158N) and a jimpy (158JP) cell line mutated for the proteolipid protein PLP/DM20. Fluorescence microscopy, flow cytometry, and western blotting analysis allow to identify major myelin proteins (PLP colocalizing with mitochondria; myelin basic protein), oligodendrocyte (CNPase and myelin oligodendrocyte glycoprotein), and peroxisomal markers [adrenoleukodystrophy protein, PMP70, acyl-CoA oxidase 1 (ACOX1), l-peroxisomal bifunctional enzyme, and catalase]. Using electron microscopy, peroxisomes were identified in the two cell lines. Gene expression (ATP-binding cassette, ABCD1, Abcd2, Abcd3, and Acox1) involved in peroxisomal transport or beta-oxidation of fatty acids was evaluated using quantitative PCR. 4-phenylbutyrate treatment increases expression of ACOX1, l-peroxisomal bifunctional enzyme, PLP, myelin oligodendrocyte glycoprotein, and CNPase, mainly in 158N cells. In both cell lines, 4-phenylbutyrate-induced ACOX1 and catalase activities while only Abcd2 gene was up-regulated in 158JP. Moreover, the higher mitochondrial activity and content observed in 158JP were associated with higher glutathione content and increased basal production of reactive oxygen species revealing different redox statuses. Altogether, 158N and 158JP cells will permit studying the relationships between peroxisomal defects, mitochondrial activity, and oligodendrocyte functions.
Johannes Berger - One of the best experts on this subject based on the ideXlab platform.
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Abcd2 Is a Strong Modifier of the Metabolic Impairments in Peritoneal Macrophages of ABCD1-Deficient Mice
2016Co-Authors: Zahid Muneer, Johannes Berger, Christoph Wiesinger, Hauke B Werner, Sonja Forss-petterAbstract:The inherited peroxisomal disorder X-linked adrenoleukodystrophy (X-ALD), associated with neurodegeneration and inflammatory cerebral demyelination, is caused by mutations in the ABCD1 gene encoding the peroxisomal ATP-binding cassette (ABC) transporter ABCD1 (ALDP). ABCD1 transports CoA-esters of very long-chain fatty acids (VLCFA) into peroxisomes for degradation by b-oxidation; thus, ABCD1 deficiency results in VLCFA accumulation. The closest homologue, ABCD2 (ALDRP), when overexpressed, compensates for ABCD1 deficiency in X-ALD fibroblasts and in ABCD1-deficient mice. Microglia/macrophages have emerged as important players in the progression of neuroinflammation. Human monocytes, lacking significant expression of ABCD2, display severely impaired VLCFA metabolism in X-ALD. Here, we used thioglycollate-elicited primary mouse peritoneal macrophages (MPMW) from ABCD1 and Abcd2 single- and double-deficient mice to establish how these mutations affect VLCFA metabolism. By quantitative RT-PCR, Abcd2 mRNA was about half as abundant as ABCD1 mRNA in wild-type and similarly abundant in ABCD1-deficient MPMW. VLCFA (C26:0) accumulated about twofold in ABCD1-deficient MPMW compared with wild-type controls, as measured by gas chromatography-mass spectrometry. In Abcd2-deficient macrophages VLCFA levels were normal. However, upon ABCD1/Abcd2 double-deficiency, VLCFA accumulation was markedly increased (sixfold) compared with ABCD1-deficient MPMW. Elovl1 mRNA, encoding the rate-limiting enzyme for elongation of VLCFA, was equally abundant across all genotypes. Peroxisomal b-oxidation of C26:
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abcd2 is a strong modifier of the metabolic impairments in peritoneal macrophages of ABCD1 deficient mice
PLOS ONE, 2014Co-Authors: Zahid Muneer, Johannes Berger, Christoph Wiesinger, Till Voigtlander, Hauke B Werner, Sonja ForsspetterAbstract:The inherited peroxisomal disorder X-linked adrenoleukodystrophy (X-ALD), associated with neurodegeneration and inflammatory cerebral demyelination, is caused by mutations in the ABCD1 gene encoding the peroxisomal ATP-binding cassette (ABC) transporter ABCD1 (ALDP). ABCD1 transports CoA-esters of very long-chain fatty acids (VLCFA) into peroxisomes for degradation by β-oxidation; thus, ABCD1 deficiency results in VLCFA accumulation. The closest homologue, ABCD2 (ALDRP), when overexpressed, compensates for ABCD1 deficiency in X-ALD fibroblasts and in ABCD1-deficient mice. Microglia/macrophages have emerged as important players in the progression of neuroinflammation. Human monocytes, lacking significant expression of ABCD2, display severely impaired VLCFA metabolism in X-ALD. Here, we used thioglycollate-elicited primary mouse peritoneal macrophages (MPMΦ) from ABCD1 and Abcd2 single- and double-deficient mice to establish how these mutations affect VLCFA metabolism. By quantitative RT-PCR, Abcd2 mRNA was about half as abundant as ABCD1 mRNA in wild-type and similarly abundant in ABCD1-deficient MPMΦ. VLCFA (C26∶0) accumulated about twofold in ABCD1-deficient MPMΦ compared with wild-type controls, as measured by gas chromatography-mass spectrometry. In Abcd2-deficient macrophages VLCFA levels were normal. However, upon ABCD1/Abcd2 double-deficiency, VLCFA accumulation was markedly increased (sixfold) compared with ABCD1-deficient MPMΦ. Elovl1 mRNA, encoding the rate-limiting enzyme for elongation of VLCFA, was equally abundant across all genotypes. Peroxisomal β-oxidation of C26∶0 amounted to 62% of wild-type activity in ABCD1-deficient MPMΦ and was significantly more impaired (29% residual activity) upon ABCD1/Abcd2 double-deficiency. Single Abcd2 deficiency did not significantly compromise β-oxidation of C26∶0. Thus, the striking accumulation of VLCFA in double-deficient MPMΦ compared with single ABCD1 deficiency was due to the loss of ABCD2-mediated, compensatory transport of VLCFA into peroxisomes. We propose that moderate endogenous expression of Abcd2 in ABCD1-deficient murine macrophages prevents the severe metabolic phenotype observed in human X-ALD monocytes, which lack appreciable expression of ABCD2. This supports upregulation of ABCD2 as a therapeutic concept in X-ALD.
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evaluation of retinoids for induction of the redundant gene abcd2 as an alternative treatment option in x linked adrenoleukodystrophy
PLOS ONE, 2014Co-Authors: Franziska D Weber, Isabelle Weinhofer, Zahid Muneer, Sonja Forsspetter, Angelika Einwich, Willi H A Weber, Harald Maier, Johannes BergerAbstract:X-linked adrenoleukodystrophy (X-ALD), the most common peroxisomal disorder, is a clinically heterogeneous disease that can manifest as devastating inflammatory cerebral demyelination (CALD) leading to death of affected males. Currently, the only curative treatment is allogeneic hematopoietic stem cell transplantation (HSCT). However, HSCT is only effective when performed at an early stage because the inflammation may progress for eighteen months after HSCT. Thus, alternative treatment options able to immediately halt the progression are urgently needed. X-ALD is caused by mutations in the ABCD1 gene, encoding the peroxisomal membrane protein ABCD1, resulting in impaired very long-chain fatty acid metabolism. The related ABCD2 protein is able to functionally compensate for ABCD1-deficiency both in vitro and in vivo. Recently, we demonstrated that of the cell types derived from CD34+ stem cells, predominantly monocytes but not lymphocytes are metabolically impaired in X-ALD. As ABCD2 is virtually not expressed in these cells, we hypothesize that a pharmacological up-regulation of ABCD2 should compensate metabolically and halt the inflammation in CALD. Retinoids are anti-inflammatory compounds known to act on ABCD2. Here, we investigated the capacity of selected retinoids for ABCD2 induction in human monocytes/macrophages. In THP-1 cells, 13-cis-retinoic acid reached the highest, fivefold, increase in ABCD2 expression. To test the efficacy of retinoids in vivo, we analyzed ABCD2 mRNA levels in blood cells isolated from acne patients receiving 13-cis-retinoic acid therapy. In treated acne patients, ABCD2 mRNA levels were comparable to pre-treatment levels in monocytes and lymphocytes. Nevertheless, when primary monocytes were in vitro differentiated into macrophages and treated with 13-cis-retinoic acid, we observed a fourfold induction of ABCD2. However, the level of ABCD2 induction obtained by retinoids alone is probably not of therapeutic relevance for X-ALD. In conclusion, our results suggest a change in promoter accessibility during macrophage differentiation allowing induction of ABCD2 by retinoids.
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impaired very long chain acyl coa β oxidation in human x linked adrenoleukodystrophy fibroblasts is a direct consequence of ABCD1 transporter dysfunction
Journal of Biological Chemistry, 2013Co-Authors: Christoph Wiesinger, Sonja Forsspetter, Guenther Regelsberger, Markus Kunze, Johannes BergerAbstract:X-linked adrenoleukodystrophy (X-ALD), an inherited peroxisomal disorder, is caused by mutations in the ABCD1 gene encoding the peroxisomal ATP-binding cassette (ABC) transporter ABCD1 (adrenoleukodystrophy protein, ALDP). Biochemically, X-ALD is characterized by an accumulation of very long-chain fatty acids and partially impaired peroxisomal β-oxidation. In this study, we used primary human fibroblasts from X-ALD and Zellweger syndrome patients to investigate the peroxisomal β-oxidation defect. Our results show that the degradation of C26:0-CoA esters is as severely impaired as degradation of unesterified very long-chain fatty acids in X-ALD and is abolished in Zellweger syndrome. Interestingly, the β-oxidation rates for both C26:0-CoA and C22:0-CoA were similarly affected, although C22:0 does not accumulate in patient fibroblasts. Furthermore, we show that the β-oxidation defect in X-ALD is directly caused by ABCD1 dysfunction as blocking ABCD1 function with a specific antibody reduced β-oxidation to levels observed in X-ALD fibroblasts. By quantification of mRNA and protein levels of the peroxisomal ABC transporters and by blocking with specific antibodies, we found that residual β-oxidation activity toward C26:0-CoA in X-ALD fibroblasts is mediated by ABCD3, although the efficacy of ABCD3 appeared to be much lower than that of ABCD1. Finally, using isolated peroxisomes, we show that β-oxidation of C26:0-CoA is independent of additional CoA but requires a cytosolic factor of >10-kDa molecular mass that is resistant to N-ethylmaleimide and heat inactivation. In conclusion, our findings in human cells suggest that, in contrast to yeast cells, very long-chain acyl-CoA esters are transported into peroxisomes by ABCD1 independently of additional synthetase activity.
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x linked adrenoleukodystrophy phenotype is independent of abcd2 genotype
Biochemical and Biophysical Research Communications, 2008Co-Authors: Esther M Maier, Muriel Asheuer, Peter U Mayerhofer, Martina Rothe, Wolfgang Kohler, Adelbert A. Roscher, Ania C. Muntau, Patrick Aubourg, Andreas Holzinger, Johannes BergerAbstract:Strikingly variable clinical phenotypes can be found in X-linked adrenoleukodystrophy (X-ALD) even with the same ABCD1 mutation. ABCD2 is the closest homolog to ABCD1. Since ABCD2 overexpression complements the loss of ABCD1 in vivo and in vitro, we have investigated the possible role of the ABCD2 gene locus as determinant of X-ALD phenotypes. Sequence and segregation analysis of the ABCD2 gene, in a large X-ALD family with different phenotypes disclosed that the identical ABCD2 alleles were inherited in brothers affected by mild (noncerebral) versus severe (childhood cerebral) X-ALD phenotypes. Moreover, two independent association studies of ABCD2 polymorphisms and clinical phenotypes showed an even allele distribution in different X-ALD phenotypes and controls. Based on these findings ABCD2 can be excluded as a major modifier locus for clinical diversity in X-ALD. These findings are of particular importance for the attempt of pharmacological induction of ABCD2 as a possible therapeutic approach in X-ALD.
Catherine Gondcaille - One of the best experts on this subject based on the ideXlab platform.
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crispr cas9 mediated knockout of ABCD1 and abcd2 genes in bv 2 cells novel microglial models for x linked adrenoleukodystrophy
Biochimica et Biophysica Acta, 2019Co-Authors: Quentin Raas, Catherine Gondcaille, Doriane Trompier, Franck Ménétrier, Yannick Hamon, Gérard Lizard, Valerio Leoni, Claudio Caccia, Stephane SavaryAbstract:X-linked adrenoleukodystrophy (X-ALD), the most frequent peroxisomal disorder, is associated with mutation in the ABCD1 gene which encodes a peroxisomal ATP-binding cassette transporter for very long-chain fatty acids (VLCFA). The biochemical hallmark of the disease is the accumulation of VLCFA. Peroxisomal defect in microglia being now considered a priming event in the pathology, we have therefore generated murine microglial cells mutated in the ABCD1 gene and its closest homolog, the Abcd2 gene. Using CRISPR/Cas9 gene editing strategy, we obtained 3 cell clones with a single or double deficiency. As expected, only the combined absence of ABCD1 and ABCD2 proteins resulted in the accumulation of VLCFA. Ultrastructural analysis by electron microscopy revealed in the double mutant cells the presence of lipid inclusions similar to those observed in brain macrophages of patients. These observations are likely related to the increased level of cholesterol and the accumulation of neutral lipids that we noticed in mutant cells. A preliminary characterization of the impact of peroxisomal defects on the expression of key microglial genes such as Trem2 suggests profound changes in microglial functions related to inflammation and phagocytosis. The expression levels of presumed modifier genes have also been found modified in mutant cells, making these novel cell lines relevant for use as in vitro models to better understand the physiopathogenesis of X-ALD and to discover new therapeutic targets.
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CRISPR/Cas9-mediated knockout of ABCD1 and Abcd2 genes in BV-2 cells: novel microglial models for X-linked Adrenoleukodystrophy
Biochimica Et Biophysica Acta-Molecular and Cell Biology of Lipids, 2019Co-Authors: Quentin Raas, Catherine Gondcaille, Doriane Trompier, Franck Ménétrier, Yannick Hamon, Gérard Lizard, Valerio Leoni, Claudio Caccia, Stephane SavaryAbstract:X-linked adrenoleukodystrophy (X-ALD), the most frequent peroxisomal disorder, is associated with mutation in the ABCD1 gene which encodes a peroxisomal ATP-binding cassette transporter for very long-chain fatty acids (VLCFA). The biochemical hallmark of the disease is the accumulation of VLCFA. Peroxisomal defect in microglia being now considered a priming event in the pathology, we have therefore generated murine microglial cells mutated in the ABCD1 gene and its closest homolog, the Abcd2 gene. Using CRISPR/Cas9 gene editing strategy, we obtained 3 cell clones with a single or double deficiency. As expected, only the combined absence of ABCD1 and ABCD2 proteins resulted in the accumulation of VLCFA. Ultrastructural analysis by electron microscopy revealed in the double mutant cells the presence of lipid inclusions similar to those observed in brain macrophages of patients. These observations are likely related to the increased level of cholesterol and the accumulation of neutral lipids that we noticed in mutant cells. A preliminary characterization of the impact of peroxisomal defects on the expression of key microglial genes such as Trem2 suggests profound changes in microglial functions related to inflammation and phagocytosis. The expression levels of presumed modifier genes have also been found modified in mutant cells, making these novel cell lines relevant for use as in vitro models to better understand the physiopathogenesis of X-ALD and to discover new therapeutic targets.
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flow cytometric analysis of the expression pattern of peroxisomal proteins ABCD1 abcd2 and abcd3 in bv 2 murine microglial cells
Methods of Molecular Biology, 2017Co-Authors: Catherine Gondcaille, Doriane Trompier, Meryam Debbabi, Thomas Nury, Imen Helali, El Mostafa Karym, Flore Geillon, Amina NajidAbstract:: Microglial cells play important roles in neurodegenerative diseases including peroxisomal leukodystrophies. The BV-2 murine immortalized cells are widely used in the context of neurodegenerative researches. It is therefore important to establish the expression pattern of peroxisomal proteins by flow cytometry in these cells. So, the expression pattern of various peroxisomal transporters (ABCD1, Abcd2, Abcd3) contributing to peroxisomal β-oxidation was evaluated on BV-2 cells by flow cytometry and complementary methods (fluorescence microscopy, and RT-qPCR). By flow cytometry a strong expression of peroxisomal proteins (ABCD1, Abcd2, Abcd3) was observed. These data were in agreement with those obtained by fluorescence microscopy (presence of numerous fluorescent dots in the cytoplasm characteristic of a peroxisomal staining pattern) and RT-qPCR (high levels of ABCD1, Abcd2, and Abcd3 mRNAs). Thus, the peroxisomal proteins (ABCD1, Abcd2, Abcd3) are expressed in BV-2 cells, and can be analyzed by flow cytometry.
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Predictive Structure and Topology of Peroxisomal ATP-Binding Cassette (ABC) Transporters
MDPI AG, 2017Co-Authors: Pierre Andreoletti, Catherine Gondcaille, Doriane Trompier, Mustapha Cherkaoui-malki, Quentin Raas, Stephane SavaryAbstract:The peroxisomal ATP-binding Cassette (ABC) transporters, which are called ABCD1, ABCD2 and ABCD3, are transmembrane proteins involved in the transport of various lipids that allow their degradation inside the organelle. Defective ABCD1 leads to the accumulation of very long-chain fatty acids and is associated with a complex and severe neurodegenerative disorder called X-linked adrenoleukodystrophy (X-ALD). Although the nucleotide-binding domain is highly conserved and characterized within the ABC transporters family, solid data are missing for the transmembrane domain (TMD) of ABCD proteins. The lack of a clear consensus on the secondary and tertiary structure of the TMDs weakens any structure-function hypothesis based on the very diverse ABCD1 mutations found in X-ALD patients. Therefore, we first reinvestigated thoroughly the structure-function data available and performed refined alignments of ABCD protein sequences. Based on the 2.85 Å resolution crystal structure of the mitochondrial ABC transporter ABCB10, here we propose a structural model of peroxisomal ABCD proteins that specifies the position of the transmembrane and coupling helices, and highlight functional motifs and putative important amino acid residues
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regulation of the adrenoleukodystrophy related gene abcd2 focus on oxysterols and lxr antagonists
Biochemical and Biophysical Research Communications, 2014Co-Authors: Doriane Trompier, Catherine Gondcaille, Gérard Lizard, Stephane SavaryAbstract:Abstract The regulation of the ABCD2 gene is recognized as a possible therapeutic target for X-linked adrenoleukodystrophy, a rare neurodegenerative disease caused by mutations in the ABCD1 gene. Up-regulation of ABCD2 expression has indeed been demonstrated to compensate for ABCD1 deficiency, restoring peroxisomal β-oxidation of very-long-chain fatty acids. Besides the known inducers of the ABCD2 gene (phenylbutyrate and histone deacetylase inhibitors, fibrates, dehydroepiandrosterone, thyroid hormone and thyromimetics), this review will focus on LXR antagonists and 22S-hydroxycholesterol, recently described as inducers of ABCD2 expression. Several LXR antagonists have been identified and their possible indication for neurodegenerative disorders will be discussed.
Thomas Langmann - One of the best experts on this subject based on the ideXlab platform.
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high glucose unsaturated and saturated fatty acids differentially regulate expression of atp binding cassette transporters abca1 and abcg1 in human macrophages
Experimental and Molecular Medicine, 2009Co-Authors: Richard Mauerer, Stefanie Ebert, Thomas LangmannAbstract:The ATP-binding cassette transporters ABCA1 and ABCG1 are highly expressed in macrophage-derived foam cells and promote reverse cholesterol efflux via biogenesis of high-density lipoproteins. The aim of this study was to analyze the direct effects of bioactive factors related to the metabolic syndrome on macrophage transcript levels of all 47 human ABC transporters. Using in vitro M-CSF predifferentiated macrophages and TaqMan low density arrays we could show that linoleic acid, palmitic acid, and high glucose levels have a major impact on ABCA1 and ABCG1 expression but do not strongly affect most other human ABC transporters. In Western blot experiments we demonstrate that ABCA1 and ABCG1 protein levels are synchronously suppressed by high glucose levels and the ω6-unsaturated fatty acid linoleic acid. We conclude that metabolites associated with the metabolic syndrome enhance the formation of atherosclerotic lesions by diminishing the reverse cholesterol transport function of ABCA1 and ABCG1.
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mapping atp binding cassette transporter gene expression profiles in melanocytes and melanoma cells
Melanoma Research, 2007Co-Authors: Susanne Heimerl, Thomas Langmann, Anja K Bosserhoff, Josef Ecker, Gerd SchmitzAbstract:ATP-binding cassette (ABC) transporters regulate the transport of a variety of physiologic substrates. Moreover, several human ABC proteins are responsible for drug exclusion in compound-treated tumor cells, providing cellular mechanisms for the development of multidrug resistance and, therefore, playing an important role in malignant transformation. As only limited information exists on the role of ABC transporters in melanoma, the aim of the study was to generate a complete expression profile of ABC transporters in this tumor entity. Using a TaqMan low-density array for 47 human ABC transporters, mRNA expression analysis was performed from normal human epidermal melanocytes (NHEM P2 and NHEM P3), nine different cell lines originating from primary melanoma (Mel Ei, Mel Juso, Mel Ho and Mel Wei), and metastases of malignant melanoma (Mel Im, Mel Ju, SK Mel 28, HTZ 19 and HMB2). Cell line-specific expression levels were compared with gene expression in pooled RNA from a variety of other human tissues. High expression levels were detected in pooled tissue RNA as well as in cells of melanocytic origin for ABCA5, ABCB2, ABCB6, ABCD3, ABCD4, ABCF1, ABCF2 and ABCF3, whereas ABCB5 revealed a melanocyte-specific high transcript level. In relation to normal melanocytes, ABCB3, ABCB6, ABCC2, ABCC4, ABCE1 and ABCF2 were significantly increased in melanoma cell lines, whereas ABCA7, ABCA12, ABCB2, ABCB4, ABCB5 and ABCD1 showed lower expression levels. In summary, we present here for the first time an ABC-transporter mRNA expression profile in melanoma in comparison to normal melanocytes. The differentially regulated ABC transporters detected by our approach may be candidate genes involved in melanoma tumorigenesis, progression and therapy resistance and could therefore be of great importance to identify novel options for melanoma therapy.
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transcriptional regulatory networks in lipid metabolism control abca1 expression
Biochimica et Biophysica Acta, 2005Co-Authors: Gerd Schmitz, Thomas LangmannAbstract:Abstract The ATP-binding cassette transporters, ABCA1 and ABCG1, are major players in mediating cellular efflux of phospholipids and cholesterol to apoA-I containing lipoproteins including preβ-HDL and αHDL and thereby exert important antiatherogenic properties. Although the exact mechanisms how ABC transporters mediate lipid transport are not completely resolved, recent evidence from several laboratories including ours suggests that vesicular transport processes involving different interactive proteins like β2-syntrophin, α1-syntrophin, Lin7, and cdc42 are critically involved in cellular lipid homeostasis controlled by ABCA1 and ABCG1. Besides sterols and fatty acids as known physiological modulators of the LXR/RXR and SREBP pathways, a growing list of natural and synthetic substances and metabolic regulators such as retinoids, PPAR-ligands, hormones, cytokines, and drugs are particularly effective in modulating ABCA1 and ABCG1 gene expression. Although ABCA1 protein amounts are regulated at the level of stability, the majority of potent activating and repressing mechanisms on ABCA1 function directly act on the ABCA1 gene promoter. Among the inducing factors, liver-X-receptors (LXR), retinoic acid receptors (RAR) and peroxisome proliferator-activated receptors (PPARs) along with their coactivators provide an amplification loop for ABCA1 and ABCG1 expression. The ABCA1 promoter is further stimulated by the ubiquitous factor Sp1 and the hypoxia-induced factor 1 (HIF1), which bind to GC-boxes and the E-box, respectively. Shutdown of ABCA1 expression in the absence of sterols or in certain tissues is mediated by corepressor complexes involving unliganded LXR, sterol-regulatory element binding protein 2 (SREBP2), Sp3, and the SCAN-domain protein ZNF202, which also impacts nuclear receptor signaling. Thus, a highly sophisticated transcriptional network controls the balanced expression of ABCA1.
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adenosine triphosphate binding cassette abc transporters are expressed and regulated during terminal keratinocyte differentiation a potential role for abca7 in epidermal lipid reorganization
Journal of Investigative Dermatology, 2003Co-Authors: Danuta Kielar, Thomas Langmann, Wolfgang E Kaminski, Gerhard Liebisch, Armin Piehler, Jurgen J Wenzel, Christoph Mohle, Susanne Heimerl, Sven O Friedrich, Alfred BottcherAbstract:Central aspects of the cellular lipid trafficking mechanisms that occur during keratinocyte differentiation are still not well understood. In the past years, evidence has accumulated to suggest that members of the superfamily of adenosine triphosphate binding cassette (ABC) transporters are critically involved in the transmembrane transport of cellular lipids. To test the hypothesis that ABC molecules are potentially involved in the epidermal transport of sphingolipids, glycerophospholipids, cholesterol, and fatty acids, we performed mRNA expression profiling of all currently known ABC molecules during in vitro differentiation of human keratinocytes and HaCaT cells. We identified six ABC molecules that displayed significant regulation during differentiation of these cells. The recently cloned transporter ABCA7 was highly expressed in keratinocytes and HaCaT cells and upregulated during differentiation. Overexpression of ABCA7 in HeLa cells resulted in increased expression of intracellular and cell surface ceramide and elevated intracellular phosphatidylserine levels. Given the observation that during terminal keratinocyte differentiation intracellular and surface ceramide levels are increased, our results render ABCA7 a candidate regulator of ceramide transport in this process. In addition to ABCA7, the cholesterol transporters ABCB1 and ABCG1 and the glutathione/glucuronide sulfate transporters ABCC1, ABCC3, and ABCC4, were strongly upregulated during keratinocyte and HaCaT cell differentiation. These findings support the notion that ABCB1 and ABCG1 are potentially implicated in cholesterol transport, whereas ABCC1, ABCC3, and ABCC4 are candidate regulators of the translocation of sulfated lipids during stratum corneum keratinization. Our results suggest specific biologic functions for members of the ABC transporter family in epidermal lipid reorganization during terminal keratinocyte differentiation.
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the zinc finger protein 202 znf202 is a transcriptional repressor of atp binding cassette transporter a1 abca1 and abcg1 gene expression and a modulator of cellular lipid efflux
Journal of Biological Chemistry, 2001Co-Authors: Mustafa Porschozcurumez, Thomas Langmann, Wolfgang E Kaminski, Susanne Heimerl, Hana Borsukova, Wolfgang Drobnik, Christian Honer, Chistoph Schumacher, Gerd SchmitzAbstract:Abstract The zinc finger gene 202 (ZNF202) located within a hypoalphalipoproteinemia susceptibility locus on chromosome 11q23 is a transcriptional repressor of various genes involved in lipid metabolism. To provide further evidence for a functional linkage between ZNF202 and hypoalphalipoproteinemia, we investigated the effect of ZNF202 expression on ATP binding cassette transporter A1 (ABCA1) and ABCG1. ABCA1 is a key regulator of the plasma high density lipoprotein pool size, whereas ABCG1 is another mediator of cellular cholesterol and phospholipid efflux in human macrophage. We demonstrate here that the full-length ZNF202m1 isoform binds to GnT repeats within the promotors of ABCA1 (−229/−210) and ABCG1 (−572/−552). ZNF202m1 expression in HepG2 cells dose-dependently repressed the promotor activities of ABCA1 and ABCG1. This transcriptional effect required the presence of the SCAN domain in ZNF202 and the functional integrity of a TATA box at position −24 of ABCA1, whereas the presence of GnT binding motifs was nonessential. The state of ZNF202 SCAN domain oligomerization affected the ability of the adjacent ZNF202 Kruppel-associated box domain to recruit the transcriptional corepressor KAP1. Overexpression of ZNF202m1 in RAW264.7 macrophages prevented the induction of ABCA1 gene expression by 20(S)OH-cholesterol and 9-cis-retinoic acid, further substantiating the interference of ZNF202 in critical elements of transcriptional activation. Finally, HDL and apoAImediated lipid efflux was significantly reduced in RAW264.7 cells stably expressing ZNF202m1. In conclusion, we have identified ABCA1 and ABCG1 as target genes for ZNF202-mediated repression and thus, provide evidence for a functional linkage between ZNF202 and hypoalphalipoproteinemia.
Doriane Trompier - One of the best experts on this subject based on the ideXlab platform.
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crispr cas9 mediated knockout of ABCD1 and abcd2 genes in bv 2 cells novel microglial models for x linked adrenoleukodystrophy
Biochimica et Biophysica Acta, 2019Co-Authors: Quentin Raas, Catherine Gondcaille, Doriane Trompier, Franck Ménétrier, Yannick Hamon, Gérard Lizard, Valerio Leoni, Claudio Caccia, Stephane SavaryAbstract:X-linked adrenoleukodystrophy (X-ALD), the most frequent peroxisomal disorder, is associated with mutation in the ABCD1 gene which encodes a peroxisomal ATP-binding cassette transporter for very long-chain fatty acids (VLCFA). The biochemical hallmark of the disease is the accumulation of VLCFA. Peroxisomal defect in microglia being now considered a priming event in the pathology, we have therefore generated murine microglial cells mutated in the ABCD1 gene and its closest homolog, the Abcd2 gene. Using CRISPR/Cas9 gene editing strategy, we obtained 3 cell clones with a single or double deficiency. As expected, only the combined absence of ABCD1 and ABCD2 proteins resulted in the accumulation of VLCFA. Ultrastructural analysis by electron microscopy revealed in the double mutant cells the presence of lipid inclusions similar to those observed in brain macrophages of patients. These observations are likely related to the increased level of cholesterol and the accumulation of neutral lipids that we noticed in mutant cells. A preliminary characterization of the impact of peroxisomal defects on the expression of key microglial genes such as Trem2 suggests profound changes in microglial functions related to inflammation and phagocytosis. The expression levels of presumed modifier genes have also been found modified in mutant cells, making these novel cell lines relevant for use as in vitro models to better understand the physiopathogenesis of X-ALD and to discover new therapeutic targets.
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CRISPR/Cas9-mediated knockout of ABCD1 and Abcd2 genes in BV-2 cells: novel microglial models for X-linked Adrenoleukodystrophy
Biochimica Et Biophysica Acta-Molecular and Cell Biology of Lipids, 2019Co-Authors: Quentin Raas, Catherine Gondcaille, Doriane Trompier, Franck Ménétrier, Yannick Hamon, Gérard Lizard, Valerio Leoni, Claudio Caccia, Stephane SavaryAbstract:X-linked adrenoleukodystrophy (X-ALD), the most frequent peroxisomal disorder, is associated with mutation in the ABCD1 gene which encodes a peroxisomal ATP-binding cassette transporter for very long-chain fatty acids (VLCFA). The biochemical hallmark of the disease is the accumulation of VLCFA. Peroxisomal defect in microglia being now considered a priming event in the pathology, we have therefore generated murine microglial cells mutated in the ABCD1 gene and its closest homolog, the Abcd2 gene. Using CRISPR/Cas9 gene editing strategy, we obtained 3 cell clones with a single or double deficiency. As expected, only the combined absence of ABCD1 and ABCD2 proteins resulted in the accumulation of VLCFA. Ultrastructural analysis by electron microscopy revealed in the double mutant cells the presence of lipid inclusions similar to those observed in brain macrophages of patients. These observations are likely related to the increased level of cholesterol and the accumulation of neutral lipids that we noticed in mutant cells. A preliminary characterization of the impact of peroxisomal defects on the expression of key microglial genes such as Trem2 suggests profound changes in microglial functions related to inflammation and phagocytosis. The expression levels of presumed modifier genes have also been found modified in mutant cells, making these novel cell lines relevant for use as in vitro models to better understand the physiopathogenesis of X-ALD and to discover new therapeutic targets.
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flow cytometric analysis of the expression pattern of peroxisomal proteins ABCD1 abcd2 and abcd3 in bv 2 murine microglial cells
Methods of Molecular Biology, 2017Co-Authors: Catherine Gondcaille, Doriane Trompier, Meryam Debbabi, Thomas Nury, Imen Helali, El Mostafa Karym, Flore Geillon, Amina NajidAbstract:: Microglial cells play important roles in neurodegenerative diseases including peroxisomal leukodystrophies. The BV-2 murine immortalized cells are widely used in the context of neurodegenerative researches. It is therefore important to establish the expression pattern of peroxisomal proteins by flow cytometry in these cells. So, the expression pattern of various peroxisomal transporters (ABCD1, Abcd2, Abcd3) contributing to peroxisomal β-oxidation was evaluated on BV-2 cells by flow cytometry and complementary methods (fluorescence microscopy, and RT-qPCR). By flow cytometry a strong expression of peroxisomal proteins (ABCD1, Abcd2, Abcd3) was observed. These data were in agreement with those obtained by fluorescence microscopy (presence of numerous fluorescent dots in the cytoplasm characteristic of a peroxisomal staining pattern) and RT-qPCR (high levels of ABCD1, Abcd2, and Abcd3 mRNAs). Thus, the peroxisomal proteins (ABCD1, Abcd2, Abcd3) are expressed in BV-2 cells, and can be analyzed by flow cytometry.
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Predictive Structure and Topology of Peroxisomal ATP-Binding Cassette (ABC) Transporters
MDPI AG, 2017Co-Authors: Pierre Andreoletti, Catherine Gondcaille, Doriane Trompier, Mustapha Cherkaoui-malki, Quentin Raas, Stephane SavaryAbstract:The peroxisomal ATP-binding Cassette (ABC) transporters, which are called ABCD1, ABCD2 and ABCD3, are transmembrane proteins involved in the transport of various lipids that allow their degradation inside the organelle. Defective ABCD1 leads to the accumulation of very long-chain fatty acids and is associated with a complex and severe neurodegenerative disorder called X-linked adrenoleukodystrophy (X-ALD). Although the nucleotide-binding domain is highly conserved and characterized within the ABC transporters family, solid data are missing for the transmembrane domain (TMD) of ABCD proteins. The lack of a clear consensus on the secondary and tertiary structure of the TMDs weakens any structure-function hypothesis based on the very diverse ABCD1 mutations found in X-ALD patients. Therefore, we first reinvestigated thoroughly the structure-function data available and performed refined alignments of ABCD protein sequences. Based on the 2.85 Å resolution crystal structure of the mitochondrial ABC transporter ABCB10, here we propose a structural model of peroxisomal ABCD proteins that specifies the position of the transmembrane and coupling helices, and highlight functional motifs and putative important amino acid residues
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regulation of the adrenoleukodystrophy related gene abcd2 focus on oxysterols and lxr antagonists
Biochemical and Biophysical Research Communications, 2014Co-Authors: Doriane Trompier, Catherine Gondcaille, Gérard Lizard, Stephane SavaryAbstract:Abstract The regulation of the ABCD2 gene is recognized as a possible therapeutic target for X-linked adrenoleukodystrophy, a rare neurodegenerative disease caused by mutations in the ABCD1 gene. Up-regulation of ABCD2 expression has indeed been demonstrated to compensate for ABCD1 deficiency, restoring peroxisomal β-oxidation of very-long-chain fatty acids. Besides the known inducers of the ABCD2 gene (phenylbutyrate and histone deacetylase inhibitors, fibrates, dehydroepiandrosterone, thyroid hormone and thyromimetics), this review will focus on LXR antagonists and 22S-hydroxycholesterol, recently described as inducers of ABCD2 expression. Several LXR antagonists have been identified and their possible indication for neurodegenerative disorders will be discussed.
