CRISPR/Cas9-mediated knockout of Abcd1 and Abcd2 genes in BV-2 cells: novel microglial models for X-linked Adrenoleukodystrophy |
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| Institution: | 1. Laboratoire Bio-PeroxIL EA7270, University of Bourgogne Franche-Comté, Dijon, France;2. Aix Marseille Univ, CNRS, INSERM, CIML, Centre d''Immunologie de Marseille-Luminy, Marseille, France;3. Laboratory of Clinical Chemistry, Hospital of Varese, ASST-Settelaghi, Milan, Italy;4. Laboratory of Medical Genetics and Neurogenetics, Foundation IRCCS Istituto Neurologico Carlo Besta, Milan, Italy;5. Centre des Sciences du Goût et de l''Alimentation, AgroSup Dijon, UMR6265/UMRA1324, CNRS, INRA, University of Bourgogne Franche-Comté, Dijon, France;6. INSERM, Dijon, France;1. Unit for Neuromuscular and Neurodegenerative Diseases, Bambino Gesù Children''s Hospital, IRCCS, Rome, Italy;2. Laboratory of Metabolomics and Proteomics, Bambino Gesù Children''s Hospital, IRCCS, Rome, Italy;3. Catalan Institution of Research and Advanced Studies (ICREA), Barcelona, Spain;4. Neurometabolic Diseases Laboratory, Bellvitge Biomedical Research Institute (IDIBELL), Hospitalet de Llobregat, Barcelona, Spain;5. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, Spain;6. Institut de Neuropatologia, Bellvitge Biomedical Research Institute (IDIBELL), Hospital Universitari de Bellvitge, Universitat de Barcelona, Spain;7. Unit of Endocrinology, Bambino Gesù Children''s Hospital, IRCCS, Rome, Italy;1. Division of Neurology, Children''s Hospital of Philadelphia, Philadelphia, PA, United States;2. DNA Diagnostic Laboratory, Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD, United States;3. The Hugo W. Moser Research Institute, Kennedy Krieger Institute Inc, Baltimore, MD, United States;4. Center for Rare Neurological Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States;5. Department of Biochemistry and Molecular Medicine, Keck School of Medicine, University of Southern California, LA, California, United States;6. MAEBIOS, Alamogordo, NM, United States;7. Save the Chimps, Fort Pierce, FL, United States;8. Department of Neuroradiology, University of California, San Francisco, Zuckerberg San Francisco General Hospital and Trauma Center, San Francisco, CA, United States;9. Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States;1. Graduate School of Biomedical Sciences, Tokushima University, Tokushima 770-8505, Japan;2. Graduate School of Technology, Industrial and Social Sciences, Tokushima University, Tokushima 770-8502, Japan;3. Diabetic Neuropathy Project, Department of Diseases and Infection, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan |
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| Abstract: | 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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