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• W2022057737 abstract "X-linked adrenoleukodystrophy is a metabolic disorder arising from a mutation/deletion in the ABCD1 gene, leading to a defect in the peroxisomal adrenoleukodystrophy protein (ALDP), which inhibits the oxidation of very long chain fatty acids (VLCFAs). Thus, these VLCFAs accumulate. In a cerebral form of ALD (cALD), VLCFA accumulation induces neuroinflammation that leads to loss of oligodendrocytes and myelin, which ultimately shortens the lifespan. To establish a relationship between the metabolic disease and inflammatory disease induction, we document that small interfering RNA (siRNA)-mediated silencing of Abcd1 (ALDP) and Abcd2 [adrenoleukodystrophy-related protein (ALDRP)] genes in mice primary astrocyte cultures resulted in accumulation of VLCFA and induction of an inflammatory response characteristic of human cALD. Correction of the metabolic defect using monoenoic FAs in Abcd1/Abcd2-silenced cultured astrocytes decreased inducible nitric oxide synthase and inflammatory cytokine expression, suggesting a link between VLCFA accumulation and inflammation. The inflammatory response was found to be mediated by transcription factors NF-κB, AP-1, and C/EBP in Abcd1/Abcd2-silenced mouse primary astrocytes. Although mechanisms of VLCFA-mediated induction of the inflammatory response have been investigated here in vitro, the in vivo mediators remain elusive. Our data represent the first study to suggest a direct link between the accumulation of VLCFA and the induction of inflammatory mediators. X-linked adrenoleukodystrophy is a metabolic disorder arising from a mutation/deletion in the ABCD1 gene, leading to a defect in the peroxisomal adrenoleukodystrophy protein (ALDP), which inhibits the oxidation of very long chain fatty acids (VLCFAs). Thus, these VLCFAs accumulate. In a cerebral form of ALD (cALD), VLCFA accumulation induces neuroinflammation that leads to loss of oligodendrocytes and myelin, which ultimately shortens the lifespan. To establish a relationship between the metabolic disease and inflammatory disease induction, we document that small interfering RNA (siRNA)-mediated silencing of Abcd1 (ALDP) and Abcd2 [adrenoleukodystrophy-related protein (ALDRP)] genes in mice primary astrocyte cultures resulted in accumulation of VLCFA and induction of an inflammatory response characteristic of human cALD. Correction of the metabolic defect using monoenoic FAs in Abcd1/Abcd2-silenced cultured astrocytes decreased inducible nitric oxide synthase and inflammatory cytokine expression, suggesting a link between VLCFA accumulation and inflammation. The inflammatory response was found to be mediated by transcription factors NF-κB, AP-1, and C/EBP in Abcd1/Abcd2-silenced mouse primary astrocytes. Although mechanisms of VLCFA-mediated induction of the inflammatory response have been investigated here in vitro, the in vivo mediators remain elusive. Our data represent the first study to suggest a direct link between the accumulation of VLCFA and the induction of inflammatory mediators. X-linked adrenoleukodystrophy (X-ALD) is a progressive peroxisomal disease characterized by deficient β-oxidation, leading to accumulation of very long chain fatty acids (VLCFAs), which ultimately affect the central nervous system (CNS) and the adrenal cortex (1Moser H.W Adrenoleukodystrophy..Curr. Opin. Neurol. 1995; 8: 221-226Crossref PubMed Scopus (18) Google Scholar, 2Moser H.W Clinical and therapeutic aspects of adrenoleukodystrophy and adrenomyeloneuropathy..J. Neuropathol. Exp. Neurol. 1995; 54: 740-745Crossref PubMed Scopus (46) Google Scholar, 3Singh I. Biochemistry of peroxisomes in health and disease..Mol. Cell. Biochem. 1997; 167: 1-29Crossref PubMed Scopus (134) Google Scholar). Different phenotypes of ALD are generally recognized, distinguished by age of onset and differences in symptoms and course of disease; these range from the rapidly progressing childhood cerebral form (cALD), to the mild adult slow-progressing adrenomyeloneuropathy (AMN), to Addison's disease (affects only the adrenal glands, not myelin), and to the asymptomatic form (or presymptomatic) (1Moser H.W Adrenoleukodystrophy..Curr. Opin. Neurol. 1995; 8: 221-226Crossref PubMed Scopus (18) Google Scholar, 2Moser H.W Clinical and therapeutic aspects of adrenoleukodystrophy and adrenomyeloneuropathy..J. Neuropathol. Exp. Neurol. 1995; 54: 740-745Crossref PubMed Scopus (46) Google Scholar, 3Singh I. Biochemistry of peroxisomes in health and disease..Mol. Cell. Biochem. 1997; 167: 1-29Crossref PubMed Scopus (134) Google Scholar). All forms of X-ALD are shown to be caused by deletion or mutations in the ABCD1 gene that belongs to the ATP binding cassette (ABC) superfamily of transmembrane transporters and encodes the adrenoleukodystrophy protein (ALDP), which is located in the peroxisomal membrane. Peroxisomes contain three additional ABC transporters: ABCD2, adrenoleukodystrophy-related protein (ALDRP); ABCD3 (PMP70, peroxisomal membrane protein); and ABCD4 (PMP70R, PMP70-related protein). Although functions of these half transporters remain unclear, they exhibit considerable sequence similarity and overlapping function(s) in peroxisomal FA metabolism, suggesting promiscuity among the functions of these transporters (4Pujol A. Ferrer I. Camps C. Metzger E. Hindelang C. Callizot N. Ruiz M. Pampols T. Giros M. Mandel J.L. Functional overlap between ABCD1 (ALD) and ABCD2 (ALDR) transporters: a therapeutic target for X-adrenoleukodystrophy..Hum. Mol. Genet. 2004; 13: 2997-3006Crossref PubMed Scopus (154) Google Scholar). The ABCD1 gene maps to chromosome Xq23 and encodes a 740-amino acid protein, ALDP. The ABCD2 gene maps to chromosome 12q11 and encodes a 741-amino acid half transporter that is 66% identical at the amino acid level with ABCD1 (5Lombard-Platet G. Savary S. Sarde C.O. Mandel J.L. Chimini G. A close relative of the adrenoleukodystrophy (ALD) gene codes for a peroxisomal protein with a specific expression pattern..Proc. Natl. Acad. Sci. USA. 1996; 93: 1265-1269Crossref PubMed Scopus (193) Google Scholar). Both ABCD1 and ABCD2 genes also share the same exon/intron structure (6Broccardo C. Troffer-Charlier N. Savary S. Mandel J.L. Chimini G. Exon organisation of the mouse gene encoding the Adrenoleukodystrophy related protein (ALDRP)..Eur. J. Hum. Genet. 1998; 6: 638-641Crossref PubMed Scopus (12) Google Scholar). The ALDRP is particularly abundant in the brain and adrenal gland (5Lombard-Platet G. Savary S. Sarde C.O. Mandel J.L. Chimini G. A close relative of the adrenoleukodystrophy (ALD) gene codes for a peroxisomal protein with a specific expression pattern..Proc. Natl. Acad. Sci. USA. 1996; 93: 1265-1269Crossref PubMed Scopus (193) Google Scholar, 7Savary S. Troffer-Charlier N. Gyapay G. Mattei M.G. Chimini G. Chromosomal localization of the adrenoleukodystrophy-related gene in man and mice..Eur. J. Hum. Genet. 1997; 5: 99-101Crossref PubMed Scopus (14) Google Scholar). Overexpression of the ALDRP in fibroblasts from X-ALD patients at least partially restores the impaired peroxisomal β-oxidation (8Netik A. Forss-Petter S. Holzinger A. Molzer B. Unterrainer G. Berger J. Adrenoleukodystrophy-related protein can compensate functionally for adrenoleukodystrophy protein deficiency (X-ALD): implications for therapy..Hum. Mol. Genet. 1999; 8: 907-913Crossref PubMed Scopus (120) Google Scholar). Furthermore, increasing expression of ALDRP by fibrates (peroxisome proliferators) in a peroxisome proliferator-activated receptor α (PPARα)-dependent fashion provides a potential therapeutic strategy for treating X-ALD (9Fourcade S. Savary S. Albet S. Gauthe D. Gondcaille C. Pineau T. Bellenger J. Bentejac M. Holzinger A. Berger J. et al.Fibrate induction of the adrenoleukodystrophy-related gene (ABCD2): promoter analysis and role of the peroxisome proliferator-activated receptor PPARalpha..Eur. J. Biochem. 2001; 268: 3490-3500Crossref PubMed Scopus (58) Google Scholar). ABCD2 expression is inducible by pharmacological agents like fibrates, and Abcd2 overexpression has been shown to compensate for ALDP deficiency in Abcd1 knockout (KO) mice, preventing VLCFA accumulation and onset of the neurological phenotype (4Pujol A. Ferrer I. Camps C. Metzger E. Hindelang C. Callizot N. Ruiz M. Pampols T. Giros M. Mandel J.L. Functional overlap between ABCD1 (ALD) and ABCD2 (ALDR) transporters: a therapeutic target for X-adrenoleukodystrophy..Hum. Mol. Genet. 2004; 13: 2997-3006Crossref PubMed Scopus (154) Google Scholar). PMP70 overexpression has been shown to partially restore VLCFA β-oxidation in X-ALD fibroblasts (10Braiterman L.T Zheng S. Watkins P.A. Geraghty M.T. Johnson G. McGuinness M.C. Moser A.B. Smith K.D. Suppression of peroxisomal membrane protein defects by peroxisomal ATP binding cassette (ABC) proteins..Hum. Mol. Genet. 1998; 7: 239-247Crossref PubMed Scopus (84) Google Scholar). Functional gene redundancy is also supported by the fact that relatively high residual VLCFA β-oxidation is present in X-ALD fibroblasts lacking ABCD1. The correction of metabolic defects after statin treatments was also suggested to be due to upregulation of ABCD2 in X-ALD cultured fibroblasts (11Singh I. Pahan K. Khan M. Lovastatin and sodium phenylacetate normalize the levels of very long chain fatty acids in skin fibroblasts of X- adrenoleukodystrophy..FEBS Lett. 1998; 426: 342-346Crossref PubMed Scopus (83) Google Scholar). These findings indicate the potential correction of the metabolic disease using pharmacogenomic approaches. Interestingly, cALD has inflammatory components, so we suggest that this ALD therapy might be beneficial for both the metabolic and the neuroinflammatory disease. VLCFA accumulation is the hallmark of X-ALD and an indication of metabolic disorder, which can subsequently lead to neuroinflammatory disease in cALD. Whether the neuroinflammatory response is directly due to abnormal VLCFA accumulation and, if so, what triggers the transition from the metabolic disorder to the severe neuroinflammatory disease, remains elusive and is the focus of ongoing research. Proinflammatory cytokines and inducible nitric oxide synthase (iNOS) have been implicated in the pathology of most brain diseases, including X-ALD, multiple sclerosis, stroke, neurotrauma, and other inflammatory and infectious neurodegenerative diseases (12Abbas N. Bednar I. Mix E. Marie S. Paterson D. Ljungberg A. Morris C. Winblad B. Nordberg A. Zhu J. Up-regulation of the inflammatory cytokines IFN-gamma and IL-12 and down-regulation of IL-4 in cerebral cortex regions of APP(SWE) transgenic mice..J. Neuroimmunol. 2002; 126: 50-57Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 13Eng L.F Ghirnikar R.S. Lee Y.L. Inflammation in EAE: role of chemokine/cytokine expression by resident and infiltrating cells..Neurochem. Res. 1996; 21: 511-525Crossref PubMed Scopus (109) Google Scholar, 14McGuinness M.C Griffin D.E. Raymond G.V. Washington C.A. Moser H.W. Smith K.D. Tumor necrosis factor-alpha and X-linked adrenoleukodystrophy..J. Neuroimmunol. 1995; 61: 161-169Abstract Full Text PDF PubMed Scopus (48) Google Scholar, 15Teixeira S.A Castro G.M. Papes F. Martins M.L. Rogerio F. Langone F. Santos L.M. Arruda P. de Nucci G. Muscara M.N Expression and activity of nitric oxide synthase isoforms in rat brain during the development of experimental allergic encephalomyelitis..Brain Res. Mol. Brain Res. 2002; 99: 17-25Crossref PubMed Scopus (26) Google Scholar, 16Lu J.F Lawler A.M. Watkins P.A. Powers J.M. Moser A.B. Moser H.W. Smith K.D. A mouse model for X-linked adrenoleukodystrophy..Proc. Natl. Acad. Sci. USA. 1997; 94: 9366-9371Crossref PubMed Scopus (219) Google Scholar, 17Stanislaus R. Singh A.K. Singh I. Lovastatin treatment decreases mononuclear cell infiltration into the CNS of Lewis rats with experimental allergic encephalomyelitis..J. Neurosci. Res. 2001; 66: 155-162Crossref PubMed Scopus (136) Google Scholar, 18Gilg A.G Singh A.K. Singh I. Inducible nitric oxide synthase in the central nervous system of patients with X-adrenoleukodystrophy..J. Neuropathol. Exp. Neurol. 2000; 59: 1063-1069Crossref PubMed Scopus (58) Google Scholar, 19Powers J.M Liu Y. Moser A.B. Moser H.W. The inflammatory myelinopathy of adreno-leukodystrophy: cells, effector molecules, and pathogenetic implications..J. Neuropathol. Exp. Neurol. 1992; 51: 630-643Crossref PubMed Scopus (221) Google Scholar). Recently, our laboratory observed decreased peroxisomal proteins and increased VLCFA in the CNS of animals with experimental autoimmune encephalomyelitis, which is an animal model of multiple sclerosis (20Singh I. Paintlia A.S. Khan M. Stanislaus R. Paintlia M.K. Haq E. Singh A.K. Contreras M.A. Impaired peroxisomal function in the central nervous system with inflammatory disease of experimental autoimmune encephalomyelitis animals and protection by lovastatin treatment..Brain Res. 2004; 1022: 1-11Crossref PubMed Scopus (55) Google Scholar). Inflammatory mediators are also reported to downregulate peroxisome function, leading to VLCFA accumulation (21Paintlia M.K Paintlia A.S. Contreras M.A. Singh I. Singh A.K. Lipopolysaccharide-induced peroxisomal dysfunction exacerbates cerebral white matter injury: attenuation by N-acetyl cysteine..Exp. Neurol. 2008; 210: 560-576Crossref PubMed Scopus (78) Google Scholar, 22Paintlia A.S Gilg A.G. Khan M. Singh A.K. Barbosa E. Singh I. Correlation of very long chain fatty acid accumulation and inflammatory disease progression in childhood X-ALD: implications for potential therapies..Neurobiol. Dis. 2003; 14: 425-439Crossref PubMed Scopus (93) Google Scholar, 23Khan M. Pahan K. Singh A.K. Singh I. Cytokine-induced accumulation of very long-chain fatty acids in rat C6 glial cells: implication for X-adrenoleukodystrophy..J. Neurochem. 1998; 71: 78-87Crossref PubMed Scopus (44) Google Scholar). In fact, VLCFA accumulation in the inflammatory region was eight to ten times greater than in the histologically normal X-ALD brain, suggesting that induction of inflammatory mediators further downregulates peroxisome function, propagating a cycle (22Paintlia A.S Gilg A.G. Khan M. Singh A.K. Barbosa E. Singh I. Correlation of very long chain fatty acid accumulation and inflammatory disease progression in childhood X-ALD: implications for potential therapies..Neurobiol. Dis. 2003; 14: 425-439Crossref PubMed Scopus (93) Google Scholar, 23Khan M. Pahan K. Singh A.K. Singh I. Cytokine-induced accumulation of very long-chain fatty acids in rat C6 glial cells: implication for X-adrenoleukodystrophy..J. Neurochem. 1998; 71: 78-87Crossref PubMed Scopus (44) Google Scholar). The Abcd1 KO mouse (X-ALD mouse) mimics the human biochemical profile, i.e., accumulation of VLCFA in tissues, decreased VLCFA β-oxidation in fibroblasts, and adrenal dysfunction (16Lu J.F Lawler A.M. Watkins P.A. Powers J.M. Moser A.B. Moser H.W. Smith K.D. A mouse model for X-linked adrenoleukodystrophy..Proc. Natl. Acad. Sci. USA. 1997; 94: 9366-9371Crossref PubMed Scopus (219) Google Scholar, 24Kobayashi T. Shinnoh N. Kondo A. Yamada T. Adrenoleukodystrophy protein-deficient mice represent abnormality of very long chain fatty acid metabolism..Biochem. Biophys. Res. Commun. 1997; 232: 631-636Crossref PubMed Scopus (154) Google Scholar, 25Forss-Petter S. Werner H. Berger J. Lassmann H. Molzer B. Schwab M.H. Bernheimer H. Zimmermann F. Nave K.A. Targeted inactivation of the X-linked adrenoleukodystrophy gene in mice..J. Neurosci. Res. 1997; 50: 829-843Crossref PubMed Scopus (166) Google Scholar). However, mice do not display the neurological phenotype until 18–20 months of age, at which time they have a mild neuropathological manifestation similar only to AMN (26Pujol A. Hindelang C. Callizot N. Bartsch U. Schachner M. Mandel J.L. Late onset neurological phenotype of the X-ALD gene inactivation in mice: a mouse model for adrenomyeloneuropathy..Hum. Mol. Genet. 2002; 11: 499-505Crossref PubMed Scopus (165) Google Scholar), which is a late-onset, slower progressing X-ALD phenotype (27Bezman L. Moser A.B. Raymond G.V. Rinaldo P. Watkins P.A. Smith K.D. Kass N.E. Moser H.W. Adrenoleukodystrophy: incidence, new mutation rate, and results of extended family screening..Ann. Neurol. 2001; 49: 512-517Crossref PubMed Scopus (266) Google Scholar). The Abcd2 KO mouse and Abcd1/Abcd2 double KO mice show evidence of oxidative stress at early ages, which is absent or minimal in the Abcd1 KO mouse (28Lu J.F Barron-Casella E. Deering R. Heinzer A.K. Moser A.B. deMesy K.L. Bentley G.S. Wand C.M. M. Pei Z. Watkins P.A. et al.The role of peroxisomal ABC transporters in the mouse adrenal gland: the loss of Abcd2 (ALDR), not Abcd1 (ALD), causes oxidative damage..Lab. Invest. 2007; 87: 261-272Crossref PubMed Scopus (28) Google Scholar). Myelin destabilization is minimal in Abcd1 KO mice, who do not have cerebral inflammation, only accelerated microglial activation in the spinal cord (29Dumser M. Bauer J. Lassmann H. Berger J. Forss-Petter S. Lack of adrenoleukodystrophy protein enhances oligodendrocyte disturbance and microglia activation in mice with combined Abcd1/Mag deficiency..Acta Neuropathol. (Berl.). 2007; 114: 573-586Crossref PubMed Scopus (26) Google Scholar). These observations clearly document that even though Abcd1 KO mice develop metabolic disease, this does not progress to neuroinflammatory disease like that observed in childhood X-ALD patients. Because deletion or mutation of the ABCD1 gene and the pathognomonic accumulation of VLCFA are used as diagnostic tools both prenatally and postnatally for X-ALD, we investigated the relationship between ABCD1/ABCD2 and the induction of neuroinflammatory disease, focusing on the effects of in vitro silencing of the Abcd1/Abcd2 genes by siRNA in mouse primary astrocyte cultures. We report that silencing of Abcd1/Abcd2 results in downregulation of peroxisomal β-oxidation activity for VLCFA and thus accumulation of VLCFA. Correction of the metabolic defect with monoenoic FAs decreased VLCFA accumulation and downregulated expression of inflammatory mediators in the Abcd1/Abcd2-silenced astrocytes. These data establish a relationship between VLCFA accumulation and increased expression of inflammatory mediators. DMEM (4.5 g/l) was purchased from Invitrogen Life Technologies; FBS and HBSS were purchased from Gibco (Invitrogen, Carlsbad, CA). ALDP and cycloxygenase-2 (COX-2) antibody were purchased from Chemicon International, Inc. (Temecula, CA). ALDRP antibody was custom-made from ANASPEC against the mouse 20-residue C-terminal sequence: 722 CKILGEDSVLKTIQTPEKTS 741. 5-Lipoxygenase (5-LOX) antibody was purchased from Cayman Chemical (Ann Arbor, MI). Oleic acid and erucic acid were purchased from Sigma-Aldrich, Inc. (Milwaukee, WI). ECL and nitrocellulose membranes were purchased from Amersham Biosciences. Fatty acid methyl ester (FAME) standards were obtained from Supelco (Bellefonte, PA). [1-14C]lignoceric acid was prepared as described earlier (30Hoshi M. Kishimoto Y. Synthesis of cerebronic acid from lignoceric acid by rat brain preparation. Some properties and distribution of the -hydroxylation system..J. Biol. Chem. 1973; 248: 4123-4130Abstract Full Text PDF PubMed Google Scholar). [1-14C]palmitic acid and 125I-labeled protein A were obtained from ICN (Cleveland, OH). C57BL6 mouse breeding pairs were purchased from Jackson Laboratory (Bar Harbor, ME) and maintained at the Medical University of South Carolina (MUSC) animal facility. All animal procedures were approved by the MUSC Animal Review Committee, and all animals received humane care in compliance with the MUSC experimental guidelines and the National Research Council's criteria for humane care (Guide for Care and Use of Laboratory Animals). Primary astrocyte-enriched cultures were prepared from the whole cortex of 1-day-old C57BL/6 mice as described earlier (31Pahan K. Sheikh F.G. Khan M. Namboodiri A.M. Singh I. Sphingomyelinase and ceramide stimulate the expression of inducible nitric-oxide synthase in rat primary astrocytes..J. Biol. Chem. 1998; 273: 2591-2600Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Briefly, the cortex was rapidly dissected in ice-cold calcium/magnesium-free HBSS at pH 7.4 as described previously (32Won J.S Choi M.R. Suh H.W. Stimulation of astrocyte-enriched culture with C2 ceramide increases proenkephalin mRNA: involvement of cAMP-response element binding protein and mitogen activated protein kinases..Brain Res. 2001; 903: 207-215Crossref PubMed Scopus (12) Google Scholar). The tissue was minced, incubated in HBSS containing trypsin (2 mg/ml) for 20 min, and washed twice in plating medium containing 10% FBS and 10 μg/ml gentamicin and then disrupted by triturating through a Pasteur pipette, after which cells were seeded in 75 cm2 culture flasks (Falcon, Franklin, NJ). After incubation at 37°C in 5% CO2 for 1 day, the medium was completely changed to the culture medium (DMEM containing 10% FBS and 10 μg/ml gentamicin). The cultures received half exchanges with fresh medium twice a week. After 10 days, the cells were shaken for at least 30 min on an orbital shaker to remove the microglia, and flasks were incubated for 1 day, after which they were shaken again for 8 h to remove the oligodendrocytes. The remaining population was used as astrocytes culture. All cultured cells were maintained at 37°C in 5% CO2. Lipids (oleic and erucic acid) were dissolved in ethanol and then diluted in DMEM for treatment of cultures. The silencer siRNA (Ambion, Austin, TX) was used for Abcd1 and/or Abcd2 silencing in primary mouse astrocytes. Briefly, mice astrocytes cultured in DMEM with 10% serum and in the presence of antibiotic were transfected with siRNA for Abcd1 and/or Abcd2 using siPORT NeoFX transfection agent (Ambion). Three siRNAs for Abcd1 and Abcd2 (Ambion) each were used (Abcd1 siRNA1, ID 162218; 5′-CCUCUACAACCUAAUUUAUtt-3′; 5′-AUAAAUUAGGUUGUAGAGGtg-3′, siRNA2, ID 60153; 5′-GGUAUUUGAAGAUGUCAAAtt-3′; 5′-UUUGACAUCUUCAAAUACCtg-3′, siRNA3, ID 60064; 5′-GGAAAUUGCCUUCUACGGGtt-3′; 5′-CCCGUAGAAGGCAAUUUCCtc-3′; Abcd2 siRNA1, ID 188185; 5′-GGCUUUAGCUUACCAGAUGtt-3′; 5′-CAUCUGGUAAGCUAAAGCCtt-3′, siRNA2, ID 214996; 5′-GGUAAAUGUCUAGAAAUGGtt-3′; 5′-CCAUUUCUAGACAUUUACCtg-3′, siRNA3, ID 214997, 5′-GCUGUAGAGAUCAAUAGAGtt-3′; 5′- CUCUAUUGAUCUCUACAGCtc-3′). The siRNAs were mixed and diluted in OPTI-MEM1 medium to a final concentration of 30 nM/well. siRNA/transfection agent was dispensed into culture plates as directed by the manufacturer. A positive control using GAPDH siRNA (Ambion) and a negative control with sequence similarity to no known human, mouse, or rat gene were included. Cells were maintained in DMEM with reduced serum (2%). Silencing was observed with Western blot and mRNA quantification. For protein analysis of the transfected cells, two wells per plate were lysed and used for protein measurements and protein levels (Western blot). Cells were maintained for 6 days in DMEM with 2% FBS before harvesting for the analysis. The peroxisomal oxidation of FAs in control, scrambled RNA (ScrRNA)-silenced, and Abcd1- and/or Abcd2-silenced astrocytes was determined in 6-well plates. β-Oxidation of FAs to acetate (water-soluble product) was determined using [1-14C]FAs as substrate [C24:0, lignoceric acid or C16:0, palmitic acid (ARC, St. Louis, MO); 150,000 dpm suspended in 0.25 mg of α-cyclodextrin/assay] (20Singh I. Paintlia A.S. Khan M. Stanislaus R. Paintlia M.K. Haq E. Singh A.K. Contreras M.A. Impaired peroxisomal function in the central nervous system with inflammatory disease of experimental autoimmune encephalomyelitis animals and protection by lovastatin treatment..Brain Res. 2004; 1022: 1-11Crossref PubMed Scopus (55) Google Scholar). Briefly, plates were washed three times with serum-free medium, and the substrate in 0.250 ml of serum-free media was added to each well. The plates were incubated for 1 h with lignoceric acid or for 30 min with palmitic acid, at 37°C. Blank wells were included in each set of plates. The reaction was stopped with 1 M KOH (0.625 ml) in methanol and the mixture was transferred to capped tubes. The methanolic solution was then incubated at 60°C for 1 h, neutralized with of 6 N HCl (0.125 ml), and partitioned with 1.25 ml of chloroform (20Singh I. Paintlia A.S. Khan M. Stanislaus R. Paintlia M.K. Haq E. Singh A.K. Contreras M.A. Impaired peroxisomal function in the central nervous system with inflammatory disease of experimental autoimmune encephalomyelitis animals and protection by lovastatin treatment..Brain Res. 2004; 1022: 1-11Crossref PubMed Scopus (55) Google Scholar). The amount of radioactivity in the upper phase represents the amount of [1-14C]FA oxidized to acetate. Cells grown in parallel in the same plate were used to determine the protein present in the assays. Experiments were performed in triplicate. Total lipids were extracted from control and treated cells as described previously (33Wilson R. Sargent J.R. Lipid and fatty acid composition of brain tissue from adrenoleukodystrophy patients..J. Neurochem. 1993; 61: 290-297Crossref PubMed Scopus (47) Google Scholar). The FAMEs were analyzed by GC (Shimadzu chromatograph GC-15A attached to a Shimadzu chromatopac C-R3A integrator) using a fused silica capillary column (25 M 007 series methyl silicone, 0.25 mm internal diameter, 0.25 μm film thickness) from Quadrex Corporation (New Haven, CT). The column temperature was programmed at 125°C for 5 min, raised to 280°C at the rate of 5°C per min, and then to 295°C, at the rate of 25°C per min. The temperature was held constant at 295°C for 5 min. The injection block was set at 200°C, and the detector was set at 320°C. The separated components were identified by comparison with retention times of standard FAME. The area under the peaks of identified FAs was set as 100%. The individual FAs were measured as area percent. The cells were washed with cold Tris-buffered saline (20 mM Trizma base and 137 mM NaCl, pH 7.5) and lysed in 1× SDS sample-loading buffer [62.5 mM Trizma base, 2% (w/v) SDS,10% glycerol], and after sonication and centrifugation at 15,000 g for 5 min, the supernatant was used for the immunoblot assay. The protein concentration of samples was determined with a detergent-compatible protein assay reagent (Bio-Rad) using BSA as the standard. The sample was boiled for 3 min with 0.1 vol of 10% β-mercaptoethanol and 0.5% bromphenol blue mix. Then 40 μg of total cellular protein was resolved by electrophoresis on 8% or 12% polyacrylamide gels, electrotransferred to a polyvinylidene difluoride filter, and blocked with Tween 20-containing TBS (TBST; 10 mM Trizma base, pH 7.4, 1% Tween 20, and 150 mM NaCl) with 5% skim milk. After incubation with antiserum raised against mice ALDP, ALDRP, COX-2, 5-LOX, and iNOS, the membranes were then incubated with HRP-conjugated anti-rabbit or mouse IgG for 1 h. The membranes were detected by autoradiography using ECL-plus (Amersham Biosciences) after washing with TBST buffer. Following total RNA extraction using TRIzol (Invitrogen) per the manufacturer's protocol, single-stranded cDNA was synthesized from total RNA. Five micrograms of total RNA was treated with 2 units of DNase I (bovine pancreas; Sigma) for 15 min at room temperature in an 18 μl vol containing 1X PCR buffer and 2 mM MgCl2. This was then inactivated by incubation with 25 mM EDTA (2 μl) at 65°C for 15 min. Next, 2 μl of random primers was added and annealed to the RNA according to the manufacturer's protocol. cDNA was synthesized in a 5 μl reaction containing 5 μg of total RNA and 50–100 units of reverse transcriptase by incubating the tubes at 42°C for 60 min. Total RNA isolation from control, ScrRNA-silenced, and Abcd1- and/or Abcd2-silenced astrocytes cultures was performed using TRIzol (Invitrogen) according to the manufacturer's protocol. Real-time PCR was conducted using Bio-Rad iCycler (iCycler iQ Multi-Color Real Time PCR Detection System; Bio-Rad). Single-stranded cDNA was synthesized from total RNA as described. The primer sets for use were designed (Oligoperfect™ designer, Invitrogen) and synthesized from Integrated DNA Technologies (Coralville, IA). The primer sequences for TNF-α: forward, 5′-ctt ctg tct act gaa ctt cgg ggt-3′ and reverse, 5′-tgg aac tga tga gag gga gcc-3′; glyceraldehyde-3-phosphate dehydrogenase: forward, 5′-cct acc ccc aat gta tcc gtt gtg-3′ and reverse, 5′-gga gga atg gga gtt gct gtt gaa-3′; IL-1β: forward, 5′-gag aga caa gca acg aca aaa tcc-3′ and reverse, 5′-ttc cca tct tct tct ttg ggt att g-3′; iNOS: forward, 5′-gga aga gga aca act act gct ggt-3′ and reverse, 5′-gaa ctg agg gta cat gct gga gc-3′; 18S: forward, 5′-gaa aac att ctt ggc aaa tgc ttt-3′ and reverse, 5′-gccgct aga ggt gaa att ctt-3′. IQ™ SYBR Green Supermix was purchased from Bio-Rad. Thermal cycling conditions were as follows: activation of DNA polymerase at 95°C for 10 min, followed by 40 cycles of amplification at 95°C for 30 s and 58.3°C for 30 s. The normalized expression of target gene with respect to glyceraldehyde-3-phosphate dehydrogenase or 18S RNA was computed for all samples using a Microsoft Excel data spreadsheet. Production of nitric oxide (NO) was determined by assaying culture supernatants for nitrite, a stable reaction product of NO and molecular oxygen. Briefly, 100 μl of culture supernatant was allowed to react with 100 μl of Griess reagent (34Dobashi K. Pahan K. Chahal A. Singh I. Modulation of endogenous antioxidant enzymes by nitric oxide in rat C6 glial cells..J. Neurochem. 1997; 68: 1896-1903Crossref PubMed Scopus (88) Google Scholar) and incubated at room temperature for 15 min. The optical density of the assay samples was measured spectrophotometrically at 570 nm. Fresh culture medium served as the blank in all experiments. Nitrite concentrations were calculated from a standard curve derived from the reaction of NaNO2 in the assay. Cells were silen" @default.
• W2022057737 created "2016-06-24" @default.
• W2022057737 creator A5032747758 @default.
• W2022057737 creator A5044613830 @default.
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• W2022057737 date "2009-01-01" @default.
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