<i>Abcd2</i> Is a Strong Modifier of the Metabolic Impairments in Peritoneal Macrophages of <i>Abcd1</i>-Deficient Mice

<div><p>The inherited peroxisomal disorder X-linked adrenoleukodystrophy (X-ALD), associated with neurodegeneration and inflammatory cerebral demyelination, is caused by mutations in the <i>ABCD1</i> 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, <i>ABCD1</i> deficiency results in VLCFA accumulation. The closest homologue, ABCD2 (ALDRP), when overexpressed, compensates for <i>ABCD1</i> deficiency in X-ALD fibroblasts and in <i>Abcd1</i>-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 <i>Abcd1</i> and <i>Abcd2</i> single- and double-deficient mice to establish how these mutations affect VLCFA metabolism. By quantitative RT-PCR, <i>Abcd2</i> mRNA was about half as abundant as <i>Abcd1</i> mRNA in wild-type and similarly abundant in <i>Abcd1</i>-deficient MPMΦ. VLCFA (C26∶0) accumulated about twofold in <i>Abcd1</i>-deficient MPMΦ compared with wild-type controls, as measured by gas chromatography-mass spectrometry. In <i>Abcd2</i>-deficient macrophages VLCFA levels were normal. However, upon <i>Abcd1</i>/<i>Abcd2</i> double-deficiency, VLCFA accumulation was markedly increased (sixfold) compared with <i>Abcd1</i>-deficient MPMΦ. <i>Elovl1</i> 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 <i>Abcd1</i>-deficient MPMΦ and was significantly more impaired (29% residual activity) upon <i>Abcd1</i>/<i>Abcd2</i> double-deficiency. Single <i>Abcd2</i> deficiency did not significantly compromise β-oxidation of C26∶0. Thus, the striking accumulation of VLCFA in double-deficient MPMΦ compared with single <i>Abcd1</i> deficiency was due to the loss of ABCD2-mediated, compensatory transport of VLCFA into peroxisomes. We propose that moderate endogenous expression of <i>Abcd2</i> in <i>Abcd1</i>-deficient murine macrophages prevents the severe metabolic phenotype observed in human X-ALD monocytes, which lack appreciable expression of <i>ABCD2</i>. This supports upregulation of <i>ABCD2</i> as a therapeutic concept in X-ALD.</p></div

A Single Lung Transplant in a Patient with Fabry Disease: Causality or Far-Fetched? A Case Report

Introduction. Fabry disease is a rare X-linked lysosomal storage disorder, characterized by an α-galactosidase A deficiency resulting in globotriaosylceramide storage within cells. Subsequently, various organ systems are involved, clinically the most important are kidneys, the heart, and the peripheral and central nervous systems. Although obstructive lung disease is a common pathological finding in Fabry disease, pulmonary involvement is a clinically disregarded feature. Case Presentation. We report a patient with a diagnosis of chronic obstructive pulmonary disease (COPD) who received a single lung transplant in 2007. Later, a kidney biopsy revealed the diagnosis of Fabry disease, which was confirmed by enzymatic and genetic testing. Ultrastructural changes in a native lung biopsy were consistent with the diagnosis. Although the association of a lung transplant and Fabry disease appears far-fetched on first sight, respiratory impairment cannot be denied in Fabry disease. Conclusion. With this case presentation, we would like to stimulate discussion about rare differential diagnoses hidden beneath widespread disease and that a correct diagnosis is the base of an optimal treatment strategy for each patient. Overall, the patient might have benefited from specific enzyme replacement therapy, especially in view of the chronic kidney disease

VLCFA levels in wild-type, <i>Abcd1</i> and <i>Abcd2</i> single-deficient and <i>Abcd1/Abcd2</i> double-deficient mouse peritoneal macrophages.

<p>The concentrations of the VLCFA species C26∶0, C24∶0 and C22∶0 and the LCFA C16∶0 were determined by GC-MS. The relative amounts of fatty acids, expressed as ratios: (A) C26∶0/C22∶0; (B) C26∶0/C16∶0; (C) C24∶0/C22∶0 and (D), C22∶0/C16∶0 were analyzed in mouse peritoneal macrophages of wild-type (WT), <i>Abcd1</i>-deficient (<i>Abcd1</i> KO), <i>Abcd2</i>-deficient (<i>Abcd2</i> KO) and <i>Abcd1/Abcd2</i> double-deficient (DOKO) mice after 5 days in culture (<i>n</i> = 3). The graphs indicate geometric means ± SD (asymmetrical). Statistically significant differences are indicated: * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001; <i>n.s</i>., no statistically significant difference.</p

Accumulation of very long-chain fatty acids does not affect mitochondrial function in adrenoleukodystrophy protein deficiency

X-linked adrenoleukodystrophy (X-ALD, OMIM 300100) is a severe inherited neurodegenerative disease, associated with the accumulation of very long-chain fatty acids (VLCFA). The recent unexpected observation that the accumulation of VLCFA in tissues of the Abcd1-deficient mouse model for X-ALD is not due to a deficiency in VLCFA degradation, led to the hypothesis that mitochondrial abnormalities might contribute to X-ALD pathology. Here, we report that in spite of substantial accumulation of VLCFA in whole muscle homogenates, normal VLCFA levels were detected in mitochondria obtained by organellar fractionation. Polarographic analyses of the respiratory chain as well as enzymatic assays of isolated muscle mitochondria revealed no differences between X-ALD and control mice. Moreover, analysis by electron microscopy, revealed normal size, structure and localization of mitochondria in muscle of both groups. Similar to the results obtained in skeletal muscle, the mitochondrial enzyme activities in brain homogenates of Abcd1-deficient and wild-type animals also did not differ. Finally, studies on mitochondrial oxidative phosphorylation in permeabilized human skin fibroblasts of X-ALD patients and controls revealed no abnormalities. Thus, we conclude that the accumulation of VLCFA per se does not cause mitochondrial abnormalities and vice versa-mitochondrial abnormalities are not responsible for the accumulation of VLCFA in X-ALD mic

Strategy and molecular evidence for targeted inactivation of the <i>Abcd2</i> gene.

<p>(A) Structure of the 5′ region of the <i>Abcd2</i> gene, the targeting construct and the disrupted gene. Exons (Ex) 1–3 are depicted as boxes with the protein coding region in gray. After homologous recombination, 1.24 kb of the <i>Abcd2</i> gene, including the transcription and translation start sites and most of exon 1, is replaced by the <i>neomycin resistance</i> gene cassette (Neo) of the targeting construct. Positions of the PCR primers (P1, P2) and the Southern blot probe (****) used in genotyping to detect homologous recombination are indicated. Restriction sites used for cloning or analysis are marked: H, <i>Hind</i>III; N, <i>Nde</i>I; Not, <i>Not</i>I; S, <i>Sca</i>I; Sp, <i>Sph</i>I and X, <i>Xho</i>I. (B) Southern blot analysis of <i>Sca</i>I-digested DNA from <i>Abcd2</i>^−/−, <i>Abcd2</i>^+/+ and <i>Abcd2</i>^+/− mice. Chemiluminescent detection of a digoxygenin-labelled probe from the 5′ flanking DNA verified homologous recombination based on deletion of a <i>Sca</i>I-site in exon 1 of <i>Abcd2</i> resulting in 3.9-kb and 4.4-kb fragments from the wild-type (WT) and knock-out (KO) alleles, respectively. (C) Northern blot analysis of mRNA from brain and skeletal muscle of adult <i>Abcd2</i>^−/− and <i>Abcd2</i>^+/+ mice. A ³²P-labelled <i>Abcd2</i> cDNA probe hybridized to the major 4.2 kb <i>Abcd2</i> mRNA and minor (5.5 and 2.8 kb) variants in both tissues of wild-type mice, but not in <i>Abcd2</i>^−/− mice. As a loading control, the blot was re-probed with β-actin cDNA, which in skeletal muscle detects a shorter, more abundant mRNA than in brain. (D) Reverse transcription-coupled PCR analysis of <i>Abcd2</i> mRNA expression in primary peritoneal macrophages from two <i>Abcd2</i>^+/+ and two <i>Abcd2</i>^−/− mice. <i>Abcd2</i>-specific primers amplified the expected 1,051-bp product in <i>Abcd2</i>^+/+ but not in <i>Abcd2</i>^−/− macrophages. As a positive control, amplification of <i>Iba1</i> cDNA (310-bp fragment) was confirmed in all samples.</p