Role of ALDP (ABCD1) and Mitochondria in X-Linked Adrenoleukodystrophy

Peroxisomes are single membrane-bound subcellular organelles present in most eukaryotic cells (8). Peroxisomes are involved in several vital metabolic pathways, including β-oxidation of very-long-chain fatty acids (VLCFA; C>22:0), plasmalogen biosynthesis, oxidation of H2O2, α-oxidation of phytanic acid, bile acid synthesis, and cholesterol biosynthesis (40). Two major classes of peroxisomal disorders have been described. The first class, peroxisomal biogenesis disorders (PBDs; McKusick 601539), is a heterogeneous group of autosomal recessive diseases characterized by alterations in various peroxisomal proteins (called peroxins and encoded by PEX genes) involved in peroxisome biogenesis (38). PBDs include Zellweger syndrome (McKusick 214100), neonatal adrenoleukodystrophy (McKusick 202370), infantile Refsum's disease (McKusick 266510), and rhizomelic chondrodysplasia punctata (McKusick 215100). The second class of peroxisomal disorders, typified by X-linked adrenoleukodystrophy (X-ALD; McKusick 300100), includes disorders with a single peroxisomal enzyme or protein defect. X-ALD is the most common peroxisomal disorder, with an incidence of approximately 1 in 17,000 (4, 9). It is a postnatal rapidly progressive disease that affects primarily the central nervous system white matter, the adrenal cortex, and the testis (23). The biochemical signature of X-ALD is increased levels of saturated unbranched VLCFA in plasma and tissues, particularly in the cholesterol ester, ganglioside, and proteolipid fractions of the brain white matter and cholesterol esters of the adrenal cortex (23). It has been clearly established that in fibroblasts, white cells, and amniocytes from X-ALD patients, there is a decrease in peroxisomal VLCFA degradation. Reduced activity of peroxisomal very-long-chain acyl coenzyme A (acyl-CoA) synthetase (VLCS), the enzyme that activates VLCFA to initiate their degradation, has been demonstrated in fibroblasts from X-ALD patients. However, the X-ALD gene, ABCD1, identified by positional cloning (24), encodes a protein (ALDP) that is a member of the ATP binding cassette (ABC) transporter superfamily of membrane proteins (16). ALDP is located in the peroxisomal membrane (25) but has no homology to any known VLCS (34) and no demonstrable VLCS activity (36). The role of ALDP in VLCFA metabolism, the pathophysiology of X-ALD, and its relationship to VLCS activity have yet to be determined. Fatty acids are activated by thioesterification to CoA by fatty acyl-CoA synthetases before they can participate in either catabolic or anabolic pathways (41). Fatty acyl-CoA synthetases capable of activating short-chain fatty acids (SCFA; C2 to C4), medium-chain fatty acids (MCFA; C6 to C10), long-chain fatty acids (LCFA; C12 to C20), or VLCFA (C>20) have been described. The protein encoded by the VLCS gene activates both VLCFA and LCFA, in contrast to long-chain acyl-CoA synthetase, which activates only LCFA. Long-chain acyl-CoA synthetase activity is found in peroxisomes, mitochondria, and microsomes, while VLCS activity is only found in peroxisomes and microsomes. Steinberg et al. (37) reported that for VLCS, the rate of activation of LCFA is 10- to 20-fold higher than the rate of activation of VLCFA. It has been suggested that ALDP is directly involved in VLCFA β-oxidation through transport of VLCS, VLCFA, or a required cofactor across the peroxisomal membrane. It should be noted, however, that the absence of ALDP results in the reduction, but not elimination, of VLCS activity and VLCFA β-oxidation in peroxisomes, suggesting either that there are compensatory activities in the peroxisome or that the effect of ALDP on peroxisomal VLCFA β-oxidation in fibroblasts is indirect. There are an estimated 48 mammalian ABC proteins (7), located in cellular and subcellular membranes, that transport a wide variety of substrates, including ions, sugars, amino acids, proteins, and lipids (15, 16). Mammalian ABC transporter proteins typically consist of two hydrophobic transmembrane domains and two hydrophilic nucleotide-binding folds encoded by a single gene. Peroxisomal ABC transporters (7) comprise a subgroup (D) of related proteins that are encoded as half-transporters with a single transmembrane domain and a single nucleotide-binding fold. In mammals, there are four ABC subfamily D (ABCD) proteins, ALDP (encoded by the ABCD1 gene), the adrenoleukodystrophy-related protein ALDRP (encoded by the ABCD2 gene), the 70-kDa peroxisomal membrane protein PMP70 (encoded by the ABCD3 gene), and the PMP70-related protein PMP70R (encoded by the ABCD4 gene). Other mammalian ABC half-transporters identified to date dimerize to form functional transporters (33, 35). Homodimerization of ALDP and heterodimerization of ALDP with ALDR and PMP70 in vitro were demonstrated in our laboratory (34) and by Liu et al. (19). The functional significance of these dimers is unknown. However, differences in substrate transport among the various possible homo- and heterodimers could reflect the metabolic demands of various cell types since the peroxisomal ABC transporters are known to have differing tissue expression patterns in vivo (3, 12, 28, 39). This laboratory (6, 17) and others (10, 27) have demonstrated that overexpression of ALDR or PMP70, as well as ALDP, cDNA in fibroblasts from X-ALD patients improves peroxisomal β-oxidation. Thus, these peroxisomal ABC half-transporters, either specifically or nonspecifically, are able to facilitate peroxisomal VLCFA β-oxidation in fibroblasts lacking ALDP. Previously, we reported the effect of a pharmacological agent, sodium 4-phenylbutyrate (4PBA), on fibroblasts from human PBD and X-ALD patients and ALD mice and showed that 4PBA treatment corrects VLCFA β-oxidation and VLCFA levels in these cells (17, 43). Initially, correction of VLCFA β-oxidation and VLCFA levels in these cells was attributed to an observed two- to threefold increase in ALDRP expression (17). However, more recent data suggest that increased histone acetylation, unrelated to increased ALDR expression, is associated with induction of VLCFA β-oxidation in cultured skin fibroblasts and indicates that induction of peroxisomal VLCFA β-oxidation by 4PBA and other pharmacological agents was associated with induction of mitochondrial LCFA β-oxidation (22). These data imply that there is intracellular communication between mitochondria and peroxisomes in human and mouse fibroblasts. Recently, Baumgart et al. reported mitochondrial alterations caused by defective peroxisomal biogenesis in a mouse model (PEX5−/−) of Zellweger syndrome (2). These included severe alterations of mitochondrial ultrastructure, changes in the expression and activity of mitochondrial respiratory chain complexes, and an increase in the heterogeneity of the mitochondrial component in various organs and specific cell types (2). Mitochondrial abnormalities have also been reported in seminal studies documenting a peroxisomal defect in Zellweger syndrome (14) and in X-ALD (30). There are precedents, therefore, for mitochondrial involvement in peroxisomal disorders. In this study, we measured peroxisomal VLCFA β-oxidation activity and VLCFA levels in ALD mouse tissues before and after treatment with various pharmacological agents to investigate the relationship between peroxisomal VLCFA β-oxidation and VLCFA levels. The relationship between mitochondrial and peroxisomal fatty acid β-oxidation activities in cultured skin fibroblasts (i) with naturally occurring mutations in fatty acid metabolism and (ii) in the presence of drugs affecting mitochondrial activity was investigated. The role of mitochondria in the induction of peroxisomal VLCFA β-oxidation in fibroblasts by 4PBA was examined, and mitochondria in mouse tissue were examined ultrastructurally for morphological abnormalities.