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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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Peroxisomal ABC transporters and X-linked adrenoleukodystrophy
médecine sciences, 2012Co-Authors: Flore Geillon, Catherine Gondcaille, Doriane Trompier, Gérard Lizard, Stephane SavaryAbstract:X-linked adrenoleukodystrophy (X-ALD) is a complex neurodegenerative disease associated with mutations in the ABCD1 gene, which encodes for a Peroxisomal ABC transporter. Thanks to the efforts of the ELA foundation and to the recent successes of gene therapy published in Science in 2009, X-ALD is better known but still remains poorly understood. The exact role of ABCD1 and its homologs, as well as the exact link between the biochemical and metabolic Peroxisomal defects and the clinical symptoms of the disease remain to be elucidated. This review summarizes the knowledge concerning the subfamily D of the ABC transporter family and concerning X-ALD, the most frequent Peroxisomal Disorder.
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induction of the adrenoleukodystrophy related gene abcd2 by thyromimetics
The Journal of Steroid Biochemistry and Molecular Biology, 2009Co-Authors: Emmanuelle C Genin, Catherine Gondcaille, Doriane Trompier, Stephane SavaryAbstract:Abstract X-linked adrenoleukodystrophy (X-ALD) is a Peroxisomal Disorder caused by mutations in the ABCD1 ( ALD ) gene. The ABCD2 gene, its closest homolog, has been shown to compensate for ABCD1 deficiency when overexpressed. We previously demonstrated that the ABCD2 promoter contains a functional thyroid hormone response element. Thyroid hormone (T3) through its receptor TRβ can induce hepatic Abcd2 expression in rodents and transiently normalize the VLCFA level in fibroblasts of Abcd1 null mice. In a therapeutic perspective, the use of selective agonists of TRβ should present the advantage to be devoid of side effects, at least concerning the cardiotoxicity associated to TRα activation. In this study, we compared the effects of T3 with those of two thyromimetics (GC-1 and CGS 23425) specific of TRβ. Using a gene reporter assay, we demonstrated that the rat Abcd2 promoter responds to the thyromimetics in a dose-dependent way similar to what is observed with T3. We then investigated the effects of 2-, 4- and 10-day treatments on the expression of ABCD2 and its paralogs ABCD3 and ABCD4 in human cell lines by RT-qPCR. Both thyromimetics trigger up-regulation of ABCD2–4 genes in HepG2 cells and X-ALD fibroblasts. Interestingly, in X-ALD fibroblasts, while T3 is associated with a transient induction of ABCD2 and ABCD3 , the treatments with thyromimetics allow the induction to be maintained until 10 days. Further in vivo experiments in Abcd1 null mice with these thyromimetics should confirm the therapeutic potentialities of these molecules.
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Induction of the adrenoleukodystrophy-related gene (ABCD2) by thyromimetics.
The Journal of steroid biochemistry and molecular biology, 2009Co-Authors: Emmanuelle C Genin, Catherine Gondcaille, Doriane Trompier, Stephane SavaryAbstract:X-linked adrenoleukodystrophy (X-ALD) is a Peroxisomal Disorder caused by mutations in the ABCD1 (ALD) gene. The ABCD2 gene, its closest homolog, has been shown to compensate for ABCD1 deficiency when overexpressed. We previously demonstrated that the ABCD2 promoter contains a functional thyroid hormone response element. Thyroid hormone (T3) through its receptor TRbeta can induce hepatic Abcd2 expression in rodents and transiently normalize the VLCFA level in fibroblasts of Abcd1 null mice. In a therapeutic perspective, the use of selective agonists of TRbeta should present the advantage to be devoid of side effects, at least concerning the cardiotoxicity associated to TRalpha activation. In this study, we compared the effects of T3 with those of two thyromimetics (GC-1 and CGS 23425) specific of TRbeta. Using a gene reporter assay, we demonstrated that the rat Abcd2 promoter responds to the thyromimetics in a dose-dependent way similar to what is observed with T3. We then investigated the effects of 2-, 4- and 10-day treatments on the expression of ABCD2 and its paralogs ABCD3 and ABCD4 in human cell lines by RT-qPCR. Both thyromimetics trigger up-regulation of ABCD2-4 genes in HepG2 cells and X-ALD fibroblasts. Interestingly, in X-ALD fibroblasts, while T3 is associated with a transient induction of ABCD2 and ABCD3, the treatments with thyromimetics allow the induction to be maintained until 10 days. Further in vivo experiments in Abcd1 null mice with these thyromimetics should confirm the therapeutic potentialities of these molecules.
Ronald J A Wanders - One of the best experts on this subject based on the ideXlab platform.
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Peroxisomal Disorders
Oxford Medicine Online, 2016Co-Authors: Bwee Tien Poll-the, Ronald J A Wanders, Hans R WaterhamAbstract:Peroxisomal Disorders represent a group of Disorders in which there is an impairment in one or more Peroxisomal functions. Clinically, a dysfunction of peroxisomes results in most cases in neurologic symptoms of varying extent ranging from severe neurologic symptoms in children to late-onset disease in adults. In most Peroxisomal Disorders there is ocular and hearing involvement in combination with a multitude of other clinical manifestations. The Peroxisomal Disorders are subdivided into two major groups: (1) the peroxisome biogenesis Disorders (PBDs), and (2) the single peroxisome enzyme deficiencies. The PBD group comprises the Zellweger spectrum Disorders (ZSDs) and rhizomelic chondrodysplasia punctate type 1 (RCDP1) whereas the single Peroxisomal enzyme deficiency group contains several different Disorders including X-linked adrenoleukodystrophy as the most frequent Disorder. Laboratory diagnosis of a Peroxisomal Disorder involves a variety of different biochemical assays in blood and urine, and should be followed up by detailed biochemical and celbiological studies in cultured fibroblasts including complementation analysis. Prenatal diagnosis is possible either by biochemical testing or by molecular analysis.
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the human Peroxisomal abc half transporter aldp functions as a homodimer and accepts acyl coa esters
The FASEB Journal, 2008Co-Authors: Carlo W T Van Roermund, Hans R Waterham, Wouter F Visser, Lodewijk Ijlst, Arno Van Cruchten, Maxim Boek, Wim Kulik, Ronald J A WandersAbstract:Peroxisomes play a major role in human cellular lipid metabolism, including the β-oxidation of fatty acids. The most frequent Peroxisomal Disorder is X-linked adrenoleukodystrophy (X-ALD), which is caused by mutations in the ABCD1 gene. The protein involved, called ABCD1, or alternatively ALDP, is a member of the ATP-binding-cassette (ABC) transporter family and is located in the Peroxisomal membrane. The biochemical hallmark of X-ALD is the accumulation of very long-chain fatty acids (VLCFAs), due to an impaired Peroxisomal β-oxidation. Although this suggests a role of ALDP in VLCFA import, no experimental evidence is available to substantiate this. In the yeast Saccharomyces cerevisiae, peroxisomes are the exclusive site of fatty acid β-oxidation. Earlier work has shown that uptake of fatty acids into peroxisomes may occur via two routes, either as free fatty acids thus requiring intraPeroxisomal activation into acyl-CoA esters or as long-chain acyl-CoA esters. The latter route involves the two peroxiso...
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Postnatal diagnosis of Peroxisomal Disorders: a biochemical approach.
Biochimie, 2003Co-Authors: Ronald J A Wanders, Joseph M. Tager, Peter G. Barth, R B H Schutgens, H. Van Den BoschAbstract:Abstract In recent years an increasing number of inherited diseases in man has been identified in which there is an impairment of one or more Peroxisomal functions. Sofar 15 different Peroxisomal Disorders have been identified which can be subdivided into three distinct groups depending upon whether there is a generalized (group A), multiple (group B) or single (group C) loss of Peroxisomal functions. In this paper we will briefly describe the functions of peroxisomes in man which are of direct relevance for the Peroxisomal Disorders known up to now. Based upon the biochemical characteristics of the different Peroxisomal Disorders, we will describe a straightforward approach for the postnatal identification of patients suspected to suffer from suffer from a Peroxisomal Disorder. Furthermore, a detailed analysis of the biochemical procedures which should be used preferably, is given.
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stability of alkyl dihydroxyacetonephosphate synthase in human control and Peroxisomal Disorder fibroblasts
Iubmb Life, 1999Co-Authors: Jan Biermann, Ronald J A Wanders, Jeannette Gootjes, Henk Van Den BoschAbstract:Alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase) is a Peroxisomal enzyme that plays a key role in ether phospholipid biosynthesis. To determine the turnover of alkyl-DHAP synthase in several Peroxisomal Disorders, pulse-chase experiments were performed. In control fibroblasts, mature alkyl-DHAP synthase displayed a half-life of 23 +/- 12 h. In Zellweger syndrome and rhizomelic chondrodysplasia punctata fibroblast cell lines, in which alkyl-DHAP synthase cannot be imported into peroxisomes, the enzyme was mainly detected in its precursor form. This precursor form showed a much shorter half-life, 5 +/- 2 h. In contrast, when the precursor protein accumulated inside the peroxisome of a particular neonatal adrenoleukodystrophy cell line in which processing does not take place, a half-life of 18 +/- 8 h, resembling that of the mature protein in controls, was observed. In a cell line from a patient with a single deficiency in the activity of alkyl-DHAP synthase, the mature form was detected and its radioactivity decreased with a half-life of 16 +/- 7 h. Collectively, these results provide an explanation for the instability of alkyl-DHAP synthase outside its target organelle. Additionally, they indicate that both the precursor and mature form of alkyl-DHAP synthase exhibit considerable intraPeroxisomal turnover.
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Diagnosis and follow-up of a case of Peroxisomal Disorder with Peroxisomal mosaicism.
Journal of Child Neurology, 1999Co-Authors: Mercedes Pineda, Ronald J A Wanders, Hugo W. Moser, Ann B. Moser, M. Girós, Frank Roels, Marc Espeel, Montserrat Ruiz, Carlos Pavía, Juan ConillAbstract:Peroxisomal Disorder phenotypes are the result of mutations that cause defective Peroxisomal assembly or alterations in the import mechanism of Peroxisomal proteins that lead to multiple peroxisoma...
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.
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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Peroxisomal ABC transporters and X-linked adrenoleukodystrophy
médecine sciences, 2012Co-Authors: Flore Geillon, Catherine Gondcaille, Doriane Trompier, Gérard Lizard, Stephane SavaryAbstract:X-linked adrenoleukodystrophy (X-ALD) is a complex neurodegenerative disease associated with mutations in the ABCD1 gene, which encodes for a Peroxisomal ABC transporter. Thanks to the efforts of the ELA foundation and to the recent successes of gene therapy published in Science in 2009, X-ALD is better known but still remains poorly understood. The exact role of ABCD1 and its homologs, as well as the exact link between the biochemical and metabolic Peroxisomal defects and the clinical symptoms of the disease remain to be elucidated. This review summarizes the knowledge concerning the subfamily D of the ABC transporter family and concerning X-ALD, the most frequent Peroxisomal Disorder.
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induction of the adrenoleukodystrophy related gene abcd2 by thyromimetics
The Journal of Steroid Biochemistry and Molecular Biology, 2009Co-Authors: Emmanuelle C Genin, Catherine Gondcaille, Doriane Trompier, Stephane SavaryAbstract:Abstract X-linked adrenoleukodystrophy (X-ALD) is a Peroxisomal Disorder caused by mutations in the ABCD1 ( ALD ) gene. The ABCD2 gene, its closest homolog, has been shown to compensate for ABCD1 deficiency when overexpressed. We previously demonstrated that the ABCD2 promoter contains a functional thyroid hormone response element. Thyroid hormone (T3) through its receptor TRβ can induce hepatic Abcd2 expression in rodents and transiently normalize the VLCFA level in fibroblasts of Abcd1 null mice. In a therapeutic perspective, the use of selective agonists of TRβ should present the advantage to be devoid of side effects, at least concerning the cardiotoxicity associated to TRα activation. In this study, we compared the effects of T3 with those of two thyromimetics (GC-1 and CGS 23425) specific of TRβ. Using a gene reporter assay, we demonstrated that the rat Abcd2 promoter responds to the thyromimetics in a dose-dependent way similar to what is observed with T3. We then investigated the effects of 2-, 4- and 10-day treatments on the expression of ABCD2 and its paralogs ABCD3 and ABCD4 in human cell lines by RT-qPCR. Both thyromimetics trigger up-regulation of ABCD2–4 genes in HepG2 cells and X-ALD fibroblasts. Interestingly, in X-ALD fibroblasts, while T3 is associated with a transient induction of ABCD2 and ABCD3 , the treatments with thyromimetics allow the induction to be maintained until 10 days. Further in vivo experiments in Abcd1 null mice with these thyromimetics should confirm the therapeutic potentialities of these molecules.
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Induction of the adrenoleukodystrophy-related gene (ABCD2) by thyromimetics.
The Journal of steroid biochemistry and molecular biology, 2009Co-Authors: Emmanuelle C Genin, Catherine Gondcaille, Doriane Trompier, Stephane SavaryAbstract:X-linked adrenoleukodystrophy (X-ALD) is a Peroxisomal Disorder caused by mutations in the ABCD1 (ALD) gene. The ABCD2 gene, its closest homolog, has been shown to compensate for ABCD1 deficiency when overexpressed. We previously demonstrated that the ABCD2 promoter contains a functional thyroid hormone response element. Thyroid hormone (T3) through its receptor TRbeta can induce hepatic Abcd2 expression in rodents and transiently normalize the VLCFA level in fibroblasts of Abcd1 null mice. In a therapeutic perspective, the use of selective agonists of TRbeta should present the advantage to be devoid of side effects, at least concerning the cardiotoxicity associated to TRalpha activation. In this study, we compared the effects of T3 with those of two thyromimetics (GC-1 and CGS 23425) specific of TRbeta. Using a gene reporter assay, we demonstrated that the rat Abcd2 promoter responds to the thyromimetics in a dose-dependent way similar to what is observed with T3. We then investigated the effects of 2-, 4- and 10-day treatments on the expression of ABCD2 and its paralogs ABCD3 and ABCD4 in human cell lines by RT-qPCR. Both thyromimetics trigger up-regulation of ABCD2-4 genes in HepG2 cells and X-ALD fibroblasts. Interestingly, in X-ALD fibroblasts, while T3 is associated with a transient induction of ABCD2 and ABCD3, the treatments with thyromimetics allow the induction to be maintained until 10 days. Further in vivo experiments in Abcd1 null mice with these thyromimetics should confirm the therapeutic potentialities of these molecules.
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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Peroxisomal ABC transporters and X-linked adrenoleukodystrophy
médecine sciences, 2012Co-Authors: Flore Geillon, Catherine Gondcaille, Doriane Trompier, Gérard Lizard, Stephane SavaryAbstract:X-linked adrenoleukodystrophy (X-ALD) is a complex neurodegenerative disease associated with mutations in the ABCD1 gene, which encodes for a Peroxisomal ABC transporter. Thanks to the efforts of the ELA foundation and to the recent successes of gene therapy published in Science in 2009, X-ALD is better known but still remains poorly understood. The exact role of ABCD1 and its homologs, as well as the exact link between the biochemical and metabolic Peroxisomal defects and the clinical symptoms of the disease remain to be elucidated. This review summarizes the knowledge concerning the subfamily D of the ABC transporter family and concerning X-ALD, the most frequent Peroxisomal Disorder.
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induction of the adrenoleukodystrophy related gene abcd2 by thyromimetics
The Journal of Steroid Biochemistry and Molecular Biology, 2009Co-Authors: Emmanuelle C Genin, Catherine Gondcaille, Doriane Trompier, Stephane SavaryAbstract:Abstract X-linked adrenoleukodystrophy (X-ALD) is a Peroxisomal Disorder caused by mutations in the ABCD1 ( ALD ) gene. The ABCD2 gene, its closest homolog, has been shown to compensate for ABCD1 deficiency when overexpressed. We previously demonstrated that the ABCD2 promoter contains a functional thyroid hormone response element. Thyroid hormone (T3) through its receptor TRβ can induce hepatic Abcd2 expression in rodents and transiently normalize the VLCFA level in fibroblasts of Abcd1 null mice. In a therapeutic perspective, the use of selective agonists of TRβ should present the advantage to be devoid of side effects, at least concerning the cardiotoxicity associated to TRα activation. In this study, we compared the effects of T3 with those of two thyromimetics (GC-1 and CGS 23425) specific of TRβ. Using a gene reporter assay, we demonstrated that the rat Abcd2 promoter responds to the thyromimetics in a dose-dependent way similar to what is observed with T3. We then investigated the effects of 2-, 4- and 10-day treatments on the expression of ABCD2 and its paralogs ABCD3 and ABCD4 in human cell lines by RT-qPCR. Both thyromimetics trigger up-regulation of ABCD2–4 genes in HepG2 cells and X-ALD fibroblasts. Interestingly, in X-ALD fibroblasts, while T3 is associated with a transient induction of ABCD2 and ABCD3 , the treatments with thyromimetics allow the induction to be maintained until 10 days. Further in vivo experiments in Abcd1 null mice with these thyromimetics should confirm the therapeutic potentialities of these molecules.
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Induction of the adrenoleukodystrophy-related gene (ABCD2) by thyromimetics.
The Journal of steroid biochemistry and molecular biology, 2009Co-Authors: Emmanuelle C Genin, Catherine Gondcaille, Doriane Trompier, Stephane SavaryAbstract:X-linked adrenoleukodystrophy (X-ALD) is a Peroxisomal Disorder caused by mutations in the ABCD1 (ALD) gene. The ABCD2 gene, its closest homolog, has been shown to compensate for ABCD1 deficiency when overexpressed. We previously demonstrated that the ABCD2 promoter contains a functional thyroid hormone response element. Thyroid hormone (T3) through its receptor TRbeta can induce hepatic Abcd2 expression in rodents and transiently normalize the VLCFA level in fibroblasts of Abcd1 null mice. In a therapeutic perspective, the use of selective agonists of TRbeta should present the advantage to be devoid of side effects, at least concerning the cardiotoxicity associated to TRalpha activation. In this study, we compared the effects of T3 with those of two thyromimetics (GC-1 and CGS 23425) specific of TRbeta. Using a gene reporter assay, we demonstrated that the rat Abcd2 promoter responds to the thyromimetics in a dose-dependent way similar to what is observed with T3. We then investigated the effects of 2-, 4- and 10-day treatments on the expression of ABCD2 and its paralogs ABCD3 and ABCD4 in human cell lines by RT-qPCR. Both thyromimetics trigger up-regulation of ABCD2-4 genes in HepG2 cells and X-ALD fibroblasts. Interestingly, in X-ALD fibroblasts, while T3 is associated with a transient induction of ABCD2 and ABCD3, the treatments with thyromimetics allow the induction to be maintained until 10 days. Further in vivo experiments in Abcd1 null mice with these thyromimetics should confirm the therapeutic potentialities of these molecules.