X-linked adrenoleukodystrophy: very long-chain fatty acid metabolism, ABC half-transporters and the complicated route to treatment

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene that encodes a peroxisomal membrane located ABC half-transporter named ALDP. Mutations in ALDP result in elevated levels of very long-chain fatty acids (VLCFA) and reduced VLCFA beta-oxidation in peroxisomes. The peroxisomal membrane harbors three additional closely related ABC half-transporters, ALDRP, PMP70 and PMP69 (PMP70R). ABC half-transporters must dimerize to form a functional full-transporter. Whether ALDP forms a homodimer or a heterodimer has not yet been resolved, but most indirect evidence favors homodimerization. The peroxisomal ABC half-transporters are functionally related. Over-expression of ALDRP can correct the biochemical defect both in X-ALD patients cells and the Abcd1 knockout mouse, providing an exciting new possibility for treatment of X-ALD patients. This paper provides an overview of current knowledge and the problems that have been encountered.     

Suppression of peroxisomal membrane protein defects by peroxisomal ATP binding cassette (ABC) proteins

X-Linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by reduced peroxisomal very long chain fatty acid (VLCFA) beta-oxidation. The X - ALD gene product (ALDP) is a peroxisomal transmembrane protein with an ATP binding cassette (ABC). ALDP and three other ABC proteins (PMP70, ALDR, P70R) localize to the peroxisomal membrane. The function of this family of peroxisomal membrane proteins is unknown. We used complementation studies to begin analysis of their role in VLCFA beta-oxidation and on the peroxisomal membrane. Expression of either ALDP or PMP70 restores VLCFA beta- oxidation in X-ALD fibroblasts, indicating overlapping functions. Their expression also restores peroxisome biogenesis in cells that are deficient in the peroxisomal membrane protein Pex2p. Thus it is likely that complex protein interactions are involved in the function and biogenesis of peroxisomal membranes that may contribute to disease heterogeneity.      

Peroxisomal very long chain fatty acid beta-oxidation activity is determined by the level of adrenodeukodystrophy protein (ALDP) expression

Impaired peroxisomal beta-oxidation of saturated very long chain fatty acids (VLCFA, >/=C22:0) results in increased VLCFA levels in the tissues and body fluids of patients with disorders of peroxisomal biogenesis (i.e., Zellweger syndrome and neonatal adrenoleukodystrophy) and single peroxisomal protein defects (i.e., X-linked adrenoleukodystrophy (X-ALD) and acyl-CoA oxidase deficiency). We show that SV40T transformation also results in impaired peroxisomal beta-oxidation and VLCFA accumulation despite the presence of abundant peroxisomes. To explore the mechanism responsible for this observation, we have examined expression of key components of peroxisomal VLCFA beta-oxidation. We found that expression of both acyl-CoA oxidase, the rate limiting enzyme of peroxisomal VLCFA beta-oxidation and the adrenoleukodystrophy protein (ALDP), the defective gene product in X-ALD, are reduced after SV40T transformation. Surprisingly, ALDP overexpression by itself restores peroxisomal VLCFA beta-oxidation in SV40T-transformed control and X-ALD cells. These results demonstrate that ALDP is a fundamental component in VLCFA peroxisomal beta-oxidation and may serve as a "gatekeeper" for VLCFA homeostasis.     

Evaluation of pharmacological induction of fatty acid beta-oxidation in X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurometabolic disorder associated with elevated levels of saturated unbranched very-long-chain fatty acids (VLCFA; C > 22:0) in plasma and tissues, and reduced VLCFA beta-oxidation in fibroblasts, white blood cells, and amniocytes from X-ALD patients. The X-ALD gene (ABCD1) at Xq28 encodes the adrenoleukodystrophy protein (ALDP) that is related to the peroxisomal ATP-binding cassette (ABCD) transmembrane half-transporter proteins. The function of ALDP is unknown and its role in VLCFA accumulation unresolved. Previously, our laboratory has shown that sodium 4-phenylbutyrate (4PBA) treatment of X-ALD fibroblasts results in increased peroxisomal VLCFA beta-oxidation activity and increased expression of the X-ALD-related protein, ALDRP, encoded by the ABCD2 gene. In this study, the effect of various pharmacological agents on VLCFA beta-oxidation in ALD mouse fibroblasts is tested. 4PBA, styrylacetate and benzyloxyacetate (structurally related to 4PBA), and trichostatin A (functionally related to 4PBA) increase both VLCFA (peroxisomal) and long-chain fatty acid [LCFA (peroxisomal and mitochondrial)] beta-oxidation. Isobutyrate, zaprinast, hydroxyurea, and 5-azacytidine had no effect on VLCFA or LCFA beta-oxidation. Lovastatin had no effect on fatty acid beta-oxidation under normal tissue culture conditions but did result in an increase in both VLCFA and LCFA beta-oxidation when ALD mouse fibroblasts were cultured in the absence of cholesterol. The effect of trichostatin A on peroxisomal VLCFA beta-oxidation is shown to be independent of an increase in ALDRP expression, suggesting that correction of the biochemical abnormality in X-ALD is not dependent on pharmacological induction of a redundant gene (ABCD2). These studies contribute to a better understanding of the role of ALDP in VLCFA accumulation and may lead to the development of more effective pharmacological therapies.     

Co-expression of mutated and normal adrenoleukodystrophy protein reduces protein function: implications for gene therapy of X-linked adrenoleukodystrophy

Inherited defects in the X-chromosomal adrenoleukodystrophy (ALD; ABCD1) gene are the genetic cause of the severe neurodegenerative disorder X-linked adrenoleukodystrophy (X-ALD). Biochemically the accumulation of very long-chain fatty acids, caused by impaired peroxisomal beta-oxidation, is the pathognomonic characteristic of the disease. Due to the X-chromosomal inheritance of X-ALD no data are available to clarify the question whether mutated adrenoleukodystrophy proteins (ALDPs) can negatively influence normal ALDP function. Here we show that restoration of beta-oxidation in X-ALD fibroblasts following transient transfection with normal ALD cDNA is more effective in ALDP-deficient fibroblasts compared with fibroblasts expressing normal amounts of mutated ALDP. Furthermore, we utilized the HeLa Tet-on system to construct a stable HeLa cell line expressing a constant level of endogenous ALDP and doxycycline-inducible levels of mutated ALDP. The induction was doxycycline dosage-dependent and the ALDP correctly localized. Interestingly, although mutated ALDP increased >6-fold in a dosage-dependent manner the total amount of ALDP (mutated and normal) remained approximately even as demonstrated by western blot and flow cytometric analyses. Thus, apparently mutated and normal ALDP compete for integration into a limited number of sites in the peroxisomal membrane. Consequently, increased amounts of mutated ALDP resulted in decreased peroxisomal beta-oxidation and accumulation of very long-chain fatty acids. These findings have direct implications on future gene therapy approaches for treatment of X-ALD, since in some patients a non-functional endogenous protein could act in a dominant negative way or displace the introduced, normal protein.     

Probing substrate-induced conformational alterations in adrenoleukodystrophy protein by proteolysis

The adrenoleukodystrophy protein (ALDP) is a half-ABC (ATP-binding cassette) transporter localized in the peroxisomal membrane. Dysfunction of this protein is the cause of the human genetic disorder X-linked adrenoleukodystrophy (X-ALD), which is characterized by accumulation of saturated, very-long-chain fatty acids (VLCFAs). This observation suggests that ALDP is involved in the metabolism of these compounds. Whether ALDP transports VLCFAs or their derivatives across the peroxisomal membrane or some cofactors essential for the efficient peroxisomal -oxidation of these fatty acids is still unknown. In this work, we used a protease-based approach to search for substrate-induced conformational alterations on ALDP. Our results suggest that ALDP is directly involved in the transport of long- and very-long-chain acyl-CoAs across the peroxisomal membrane.     

Identification of a fourth half ABC transporter in the human peroxisomal membrane

Three half ATP-binding cassette transporters (ALDP, ALDR, PMP70) are known to be present in the human peroxisome membrane. Mutations in the gene encoding ALDP cause X-linked adrenoleukodystrophy; the role of ALDR and PMP70 in human disease is unclear. We report the cloning and characterization of a fourth human gene encoding a peroxisomal half ABC transporter. The gene, designated P70R, maps to chromosome 14q24, encodes a 73 kDa transporter most similar to PMP70, and is expressed in all human tissues examined. Because half ABC transporters heterodimerize to form functional transporters, the identification of a fourth member of this family in the peroxisome membrane has implications for our understanding of mammalian peroxisomes and the genetic disorders of peroxisomal function.      

Elongation of very long-chain fatty acids is enhanced in X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a progressive neurodegenerative disorder characterized by the accumulation of saturated and mono-unsaturated very long-chain fatty acids (VLCFA) and reduced peroxisomal VLCFA beta-oxidation activity. In this study, we investigated the role of VLCFA biosynthesis in X-ALD fibroblasts. Our data demonstrate that elongation of both saturated and mono-unsaturated VLCFAs is enhanced in fibroblasts from patients with peroxisomal beta-oxidation defects including X-ALD, and peroxisome biogenesis disorders. These data indicate that enhanced VLCFA elongation is a general phenomenon associated with an impairment in peroxisomal beta-oxidation, and not specific for X-ALD alone. Analysis of plasma samples from patients with X-ALD and different peroxisomal beta-oxidation deficiencies revealed increased concentrations of VLCFAs up to 32 carbons. We infer that enhanced elongation does not result from impaired peroxisomal beta-oxidation alone, but is due to the additional effect of unchecked chain elongation. We demonstrate that elongated VLCFAs are incorporated into complex lipids. The role of chain elongation was also studied retrospectively in samples from patients with X-ALD previously treated with "Lorenzo's oil." We found that the decrease in plasma C26:0 previously found is offset by the increase of mono-unsaturated VLCFAs, not measured previously during the trial. We conclude that evaluation of treatment protocols for disorders of peroxisomal beta-oxidation making use of plasma samples should include the measurement of saturated and unsaturated VLCFAs of chain lengths above 26 carbon atoms. We also conclude that chain elongation offers an interesting target to be studied as a possible mode of treatment for X-ALD and other peroxisomal beta-oxidation disorders.     

A very long-chain acyl-CoA synthetase-deficient mouse and its relevance to X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative and endocrine disorder resulting from mutations in ABCD1 which encodes a peroxisomal membrane protein in the ATP binding cassette superfamily. The biochemical signature of X-ALD is increased levels of saturated very long-chain fatty acids (VLCFA; carbon chains of 22 or more) in tissues and plasma that has been associated with decreased peroxisomal very long-chain acyl-CoA synthetase (VLCS) activity and decreased peroxisomal VLCFA beta-oxidation. It has been hypothesized that ABCD1, which has no demonstrable VLCS activity itself, has an indirect effect on peroxisomal VLCS activity and VLCFA beta-oxidation by transporting fatty acid substrates, VLCS protein or some required co-factor into peroxisomes. Here we report the characterization of a Vlcs knockout mouse that exhibits decreased peroxisomal VLCS activity and VLCFA beta-oxidation but does not accumulate VLCFA. The XALD/Vlcs double knockout mouse has the biochemical abnormalities observed in the individual knockout mice but does not display a more severe X-ALD phenotype. These data lead us to conclude that (1) VLCFA levels are independent of peroxisomal fatty acid beta-oxidation, (2) there is no ABCD1/VLCS interaction and (3) the common severe forms of X-ALD cannot be modeled by decreasing peroxisomal VLCS activity in the XALD mouse.     

X-linked adrenoleukodystrophy: Phenotype distribution and expression of ALDP in Spanish kindreds

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder caused by an impairment in peroxisomal β-oxidation of very long straight-chain fatty acids (VLCFAs). Six clinical phenotypes have been delineated: childhood cerebral (CCALD), adolescent cerebral (AdolCALD), adult cerebral (ACALD), adrenomyeloneuropathy (AMN), Addison-only (AO), and presymptomatic (PALD). The distribution of phenotypes varies in different countries. We have diagnosed biochemically 60 X-ALD Spanish patients belonging to 48 kindreds. Their phenotypic distribution was: CCALD plus AdolCALD, 33%; ACALD, 16%; AMN, 27%; AO, 12%; and PALD, 12%. These results contrast with the distribution described in other countries, due to a higher prevalence of the ACALD form. Regarding the expression of the protein product (ALDP), we studied 17 kindreds using immunochemical techniques and found absence of ALDP in 84% of cases. We also studied 13 females from 7 negative ALDP kindreds in order to correlate ALDP expression and the carrier status established by VLCFA measurement. In one case with normal VLCFA levels in serum and fibroblasts, we observed mosaicism in ALDP expression. This fact supports the use of this technique for identifying carriers. Am. J. Med. Genet. 76:424–427, 1998. © 1998 Wiley-Liss, Inc.     

The gene responsible for adrenoleukodystrophy encodes a peroxisomal membrane protein

Adrenoleukodystrophy is a severe genetic demye-llnating diseaseassociated with an impairment of ß-oxidation of verylong chain fatty acids (VLCFA) In peroxisomes. Earlier studieshad suggested that a deficiency in VLCFA CoA synthetase wasthe primary defect. A candidate adrenoleukodystrophy gene hasrecently been cloned and was found unexpectedly to encode aputative ATP-binding cassette transporter. We have raised monoclonalantibodies against this protein, that detect a 75kDa band. Thisprotein was absent in several patients with adrenoleukodystrophy.Immunofluorescence and Immunoelectron microscopy showed thatthe adrenoleukodystrophy protein (ALDP) is associated with theperoxisomal membrane. Distinct Immunofluorescence patterns wereobserved in cell lines from patients with Zellweger syndrome(a peroxisomal biogenesis disorder) belonging to different complementationgroups.     

Biochemical Aspects of X‐Linked Adrenoleukodystrophy

X‐linked adrenoleukodystrophy (X‐ALD) is the most common peroxisomal disorder. The disease is characterized by the accumulation of very long‐chain fatty acids (VLCFA; >C22) in plasma and tissues. X‐ALD is caused by mutations in the ABCD1 gene encoding ALDP, an adenosine triphosphate (ATP)‐binding‐cassette (ABC) transporter located in the peroxisomal membrane. In this paper, we describe the current knowledge on the function of ALDP, its role in peroxisomal VLCFA beta‐oxidation and the consequences of a defect in ALDP on VLCFA metabolism. Furthermore, we pay special attention to the role of the VLCFA elongation system in VLCFA homeostasis, with elongation of very long‐chain fatty acids like‐1 (ELOVL1) as key player, and its relevance to X‐ALD.     

Accurate DNA-based diagnostic and carrier testing for X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy is a serious and often fatal disorder, affecting the white matter of the nervous system, the adrenal cortex, and the testis. The gene mutated in X-ALD encodes a peroxisomal membrane protein, ALDP. The presence of very long chain fatty acids in plasma is highly diagnostic for affected males and carrier females, but exclusion of carrier status biochemically is unreliable. Molecular analysis of the X-ALD gene has the potential to either identify or rule out carrier status accurately, but is complicated by the existence of autosomal paralogs. We have developed and validated a robust DNA diagnostic test for this disorder involving nonnested genomic amplification of the X-ALD gene, followed by fluorescent dye-primer sequencing and analysis. This protocol provides a highly reliable means of determining carrier status in women at risk for transmitting X-ALD and is applicable to a clinical diagnostic laboratory.     

Cerebral inflammation in X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurodegenerative disease that affects approximately 1 in 25 000 males. It is characterized by elevated levels of saturated very long chain fatty acids (VLCFA), i.e., >C22:0, particularly in ganglioside and cholesterol ester fractions of brain white matter and adrenal cortex. Failure of peroxisomal very long chain fatty acyl-CoA synthetase (VLCS) to activate these VLCFA prevents their degradation by peroxisomal beta-oxidation. X-ALD maps to Xq28 and the gene encodes a peroxisomal membrane protein and not the gene for VLCS. The two most common forms of X-ALD are the cerebral (CER) form, with an inflammatory demyelinating reaction that resembles multiple sclerosis (MS), and adrenomyeloneuropathy (AMN), which involves the spinal cord and in which the inflammatory reaction is mild or absent. Investigations into the nature of the cerebral inflammatory demyelinating reaction in X-ALD will be the subject of this review.     

X-linked adrenoleukodystrophy: role of very long-chain acyl-CoA synthetases

The principal biochemical abnormality in the neurodegenerative disorder X-linked adrenoleukodystrophy (X-ALD) is elevated plasma and tissue levels of very long-chain fatty acids (VLCFA). Enzymes with very long-chain acyl-CoA synthetase (VLACS) activity are required for VLCFA metabolism, including degradation by peroxisomal beta-oxidation or incorporation into complex lipids, and may also participate in VLCFA synthesis. Two enzymes with VLACS activity, ACSVL1 and BG1, were investigated for their potential role in X-ALD biochemical pathology. Skin fibroblast mRNA levels for ACSVL1, an enzyme previously shown to be in peroxisomes and to participate in VLCFA beta-oxidation, were not significantly different between normal controls, patients with childhood cerebral X-ALD, and patients with adrenomyeloneuropathy. Similar results were obtained with mRNA for BG1, a non-peroxisomal enzyme that is highly expressed in nervous system, adrenal gland, and testis, the principal tissues pathologically affected in X-ALD. No significant differences in the immunohistochemical staining patterns of tissues expressing either ACSVL1 or BG1 were observed when wild-type and X-ALD mice were compared. Western blot analysis of BG1 protein levels showed no differences between fibroblasts from controls, cerebral X-ALD, or adrenomyeloneuropathy patients. BG1 protein levels were similar in wild-type and X-ALD mouse brain, spinal cord, testis, and adrenal gland. We hypothesized that one function of BG1 was to direct VLCFA into the cholesterol ester synthesis pathway. However, BG1 depletion in Neuro2a cells using RNA interference did not decrease incorporation of labeled VLCFA into cholesterol esters. We conclude that the role, if any, of ACSVL1 and BG1 in X-ALD biochemical pathology is indirect.     

Late onset neurological phenotype of the X-ALD gene inactivation in mice: a mouse model for adrenomyeloneuropathy

Adrenomyeloneuropathy (AMN) and cerebral childhood adrenoleukodystrophy (CCALD) are the main phenotypic variants of an X-linked inherited metabolic disorder causing demyelination, X-linked adrenoleukodystrophy (X-ALD). It is caused by mutations in the ABCD1 (ALD) gene encoding a peroxisomal ABC transporter. Inactivation of the murine ALD gene does not lead to a detectable clinical phenotype in mice up to 6 months, and no cerebral pathology resembling the childhood form (CCALD) was observed. In this work, we show that older ALD-deficient mice exhibit an abnormal neurological and behavioral phenotype, starting at around 15 months. This is correlated with slower nerve conduction, and with myelin and axonal anomalies detectable in the spinal cord and sciatic nerve, but not in brain. The phenotype of ALD-deficient mice mimics features of human AMN, thus providing a model for investigating the pathogenesis of this disease.     

The peroxisomal ABC transporter family

This review describes the current state of knowledge about the ABCD family of peroxisomal half adenosine-triphosphate-binding cassette (ABC) transporters. ABCDs are predicted to be present in a variety of eukaryotic organisms, although at present, only ABCDs in the yeast Saccharomyces cerevisiae, the plant Arabidopsis thaliana, and different mammalian species have been identified and characterized to any significant extent. The functional role of none of these ABCDs has been established definitively and awaits successful reconstitution of ABCDs, either as homo- or heterodimers into liposomes, followed by transport studies. Data obtained in S. cerevisiae suggest that the two ABCDs, which have been identified in this organism, form a heterodimer, which actually transports acyl coenzyme A esters across the peroxisomal membrane. In mammals, four ABCDs have been identified, of which one [adrenoleukodystrophy protein (ALDP)] has been implicated in the transport of the coenzyme A esters of very-long-chain fatty acids. Mutations in the gene (ABCD1) encoding ALDP are the cause of a severe X-linked disease, called X-linked adrenoleukodystrophy. The availability of mutant mice in which Abcd1, Abcd2, or Abcd3 have been disrupted will help to resolve the true role of the peroxisomal half-ABC transporters.     

Characterization of a partial pseudogene homologous to the adrenoleukodystrophy gene and application to mutation detection

The gene for the most common peroxisomal disorder, X-linked adrenoleukodystrophy (X-ALD, McKusick #300100), encodes a peroxisomal membrane transporter protein (ALDP), and comprises 10 exons spanning approximately 21 kb. So far, however, the mutation analysis at the genomic level was handicapped by the coamplification, in PCR reactions, of sequences related to the distal exons, also detected by Southern blot hybridization on genomic DNA. We isolated one clone from a human genomic phage library, which represents a partial ALD pseudogene, spanning exon 7 to exon 10 and exhibits approximately 93% sequence homology with the ALD gene in this region. Primers designed in the region of maximum mismatch between the pseudogene and the functional gene allowed the amplification of the functional exons without any contaminating sequences of the pseudogene or other related sequences. This information will greatly facilitate the detection of mutations in distal exons of the ALD gene and increase the reliability of the mutation analysis. © 1996 Wiley-Liss, Inc.