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.     

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.     

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.     

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.     

Determination of 30 X-linked adrenoleukodystrophy mutations, including 15 not previously described

X-linked Adrenoleukodystrophy (X-ALD) is the most frequent peroxisomal disease. It mainly involves the nervous system white matter, adrenal cortex and testes. Several distinct clinical phenotypes are known. The principal biochemical abnormality is the accumulation of saturated very-long-chain fatty acids (VLCFAs : > C22:0, mainly C26:0), which is due to impaired capacity for beta-oxidation in peroxisomes. Diagnosis is usually based on the VLCFA levels in plasma or cultured skin fibroblasts in both patients and carriers. In 0.1% of affected males, however, the plasma C26:0 level is borderline normal, and 15% of obligate female carriers have normal results. Effective mutation detection in these families is therefore fundamental to unambiguously determine the genetic status of each individual at risk. Of particular concern are female members of kindreds segregating X-ALD mutations, because normal VLCFA levels do not guarantee lack of carrier status. We describe a fast method for detection of X-ALD mutations. The method is based on SSCP analysis of nested PCR fragments followed by sequence-determination reactions. Using this methodology we have found X-ALD mutations in 30 kindreds, including 15 not previously reported.     

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 mice.     

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.     

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.     

Analysis of very long-chain fatty acids using electrospray ionization mass spectrometry

Elevated levels of very long-chain fatty acids (VLCFA) in plasma and tissues are the biochemical hallmark for patients with X-linked adrenoleukodystrophy (X-ALD). Current methods for the determination of VLCFA levels are laborious and time-consuming. We describe a rapid and easy method using electrospray ionization mass spectrometry (ESI-MS) with deuterated internal standards. VLCFA are hydrolyzed, extracted, and quantified in less than 4h. This includes 2h of hydrolysis and 4min of quantification. We validated the method by analyzing 60 plasma samples from controls and patients with X-ALD or Zellweger syndrome using both the ESI-MS protocol and an established method for VLCFA analysis using gas chromatography (GC). The C26:0 concentrations determined with ESI-MS in plasma and fibroblasts of X-ALD patients are in good agreement with those reported previously for GC and GC-MS. Besides saturated straight chain VLCFA, we also determined the concentrations of the mono-unsaturated VLCFA C24:1 and C26:1 and established that while C24:1 levels are not elevated, C26:1 levels are elevated in both plasma and fibroblasts from X-ALD patients.     

Early oxidative damage underlying neurodegeneration in X-adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a fatal neurodegenerative disorder, characterized by progressive cerebral demyelination cerebral childhood adrenoleukodystrophy (CCALD) or spinal cord neurodegeneration (adrenomyeloneuropathy, AMN), adrenal insufficiency and accumulation of very long-chain fatty acids (VLCFA) in tissues. The disease is caused by mutations in the ABCD1 gene, which encodes a peroxisomal transporter that plays a role in the import of VLCFA or VLCFA-CoA into peroxisomes. The Abcd1 knockout mice develop a spinal cord disease that mimics AMN in adult patients, with late onset at 20 months of age. The mechanisms underlying cerebral demyelination or axonal degeneration in spinal cord are unknown. Here, we present evidence by gas chromatography/mass spectrometry that malonaldehyde-lysine, a consequence of lipoxidative damage to proteins, accumulates in the spinal cord of Abcd1 knockout mice as early as 3.5 months of age. At 12 months, Abcd1- mice accumulate additional proteins modified by oxidative damage arising from metal-catalyzed oxidation and glycoxidation/lipoxidation. While we show that VLCFA excess activates enzymatic antioxidant defenses at the protein expression levels, both in neural tissue, in ex vivo organotypic spinal cord slices from Abcd1- mice, and in human ALD fibroblasts, we also demonstrate that the loss of Abcd1 gene function hampers oxidative stress homeostasis. We find that the alpha-tocopherol analog Trolox is able to reverse oxidative lesions in vitro, thus providing therapeutic hope. These results pave the way for the identification of therapeutic targets that could reverse the deregulated response to oxidative stress in X-ALD.     

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.     

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.      

Decreased expression of ABCD4 and BG1 genes early in the pathogenesis of X-linked adrenoleukodystrophy

Childhood cerebral adrenoleukodystrophy (CCER), adrenomyeloneuropathy (AMN) and AMN with cerebral demyelination (AMN-C) are the main phenotypic variants of X-linked adrenoleukodystrophy (ALD). It is caused by mutations in the ABCD1 gene encoding a half-size peroxisomal transporter that has to dimerize to become functional. The biochemical hallmark of ALD is the accumulation of very-long-chain fatty acids (VLCFA) in plasma and tissues. However, there is no correlation between the ALD phenotype and the ABCD1 gene mutations or the accumulation of VLCFA in plasma and fibroblast from ALD patients. The absence of genotype-phenotype correlation suggests the existence of modifier genes. To elucidate the mechanisms underlying the phenotypic variability of ALD, we studied the expression of ABCD1, three other peroxisomal transporter genes of the same family (ABCD2, ABCD3 and ABCD4) and two VLCFA synthetase genes (VLCS and BG1) involved in VLCFA metabolism, as well as the VLCFA concentrations in the normal white matter (WM) from ALD patients with CCER, AMN-C and AMN phenotypes. This study shows that: (1) ABCD1 gene mutations leading to truncated ALD protein are unlikely to cause variation in the ALD phenotype; (2) accumulation of saturated VLCFA in normal-appearing WM correlates with ALD phenotype and (3) expression of the ABCD4 and BG1, but not of the ABCD2, ABCD3 and VLCS genes, tends to be correlated with the severity of the disease, acting early in the pathogenesis of ALD.     

X-linked adrenoleukodystrophy: ABCD1 de novo mutations and mosaicism

X-linked adrenoleukodystrophy (X-ALD) is a progressive peroxisomal disorder affecting adrenal glands, testes and myelin stability that is caused by mutations in the ABCD1 (NM_000033) gene. Males with X-ALD may be diagnosed by the demonstration of elevated very long chain fatty acid (VLCFA) levels in plasma. In contrast, only 80% of female carriers have elevated plasma VLCFA; therefore targeted mutation analysis is the most effective means for carrier detection. Amongst 489 X-ALD families tested at Kennedy Krieger Institute, we identified 20 cases in which the ABCD1 mutation was de novo in the index case, indicating that the mutation arose in the maternal germ line and supporting a new mutation rate of at least 4.1% for this group. In addition, we identified 10 cases in which a de novo mutation arose in the mother or the grandmother of the index case. In two of these cases studies indicated that the mothers were low level gonosomal mosaics. In a third case biochemical, molecular and pedigree analysis indicated the mother was a gonadal mosaic. To the best of our knowledge mosaicism has not been previously reported in X-ALD. In addition, we identified one pedigree in which the maternal grandfather was mosaic for the familial ABCD1 mutation. Less than 1% of our patient population had evidence of gonadal or gonosomal mosaicism, suggesting it is a rare occurrence for this gene and its associated disorders. However, the residual maternal risk for having additional ovum carrying the mutant allele identified in an index case that appears to have a de novo mutation is at least 13%.     

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.     

Neonatal adrenoleukodystrophy: new cases, biochemical studies, and differentiation from Zellweger and related peroxisomal polydystrophy syndromes

Eight new cases of autopsy-confirmed or suspected neonatal adrenoleukodystrophy (NALD) are presented together with new biochemical data on very-long-chain fatty acids (VLCFA) and plasmalogens and a review of all previously published cases. The clinical, biochemical, and histopathologic abnormalities characteristic of this newly recognized form of adrenoleukodystrophy are analyzed in detail and compared to the principal characteristics of the similar disorder, the cerebrohepatorenal syndrome of Zellweger (ZS). Using strict pathologic criteria for the diagnosis of NALD, we find that, despite many clinical resemblances, NALD and the ZS are distinguishable on the basis of histology and peroxisomal biochemistry. Patients with NALD demonstrate adrenal atrophy, systemic infiltration by abnormal lipid-laden macrophages, and elevations of saturated VLCFA. In contrast, patients with ZS have chondrodysplasia, glomerulocystic disease of the kidney, central nervous system dysmyelination, and elevations of unsaturated as well as saturated VLCFA, but they lack adrenal atrophy. We conclude that NALD and the ZS probably represent at least two different genetic defects.     

Toxic effects of X-linked adrenoleukodystrophy-associated, very long chain fatty acids on glial cells and neurons from rat hippocampus in culture

Saturated very long chain fatty acids (VLCFAs; > or =C22:0) accumulate in X-linked adrenoleukodystrophy (X-ALD, OMIM 300100), a severe hereditary neurodegenerative disease, due to peroxisomal impairment. Previous studies analysed the development of X-ALD in humans and gene knockout animal models. However, the toxic effect of VLCFA leading to severe symptoms with progressive and multifocal demyelination, adrenal insufficiency and inflammation still remains unclear. To understand the toxic effects of VLCFA in the brain, here we exposed neural cells to VLCFA and analysed the cellular consequences. We found that oligodendrocytes and astrocytes challenged with docosanoic- (C22:0), tetracosanoic- (C24:0) and hexacosanoic acids (C24:0) die within 24 h. VLCFA-induced depolarization of mitochondria in situ and increased intracellular Ca2+ level in all three brain cell types provides indications about the mechanism of toxicity of VLCFA. Interestingly, VLCFAs affect to the largest degree the myelin-producing oligodendrocytes. In isolated mitochondria, VLCFAs exert a detrimental effect by affecting the inner mitochondrial membrane and promoting the permeability transition. In conclusion, we suggest that there is a potent toxic activity of VLCFA due to dramatic cell physiological effects with mitochondrial dysfunction and Ca2+ deregulation. This provides the first evidence for mitochondrial-based cell death mechanisms in neurodegenerative disease with peroxisomal defects and subsequent VLCFA accumulation.     

肾上腺脑白质营养不良外显子1,5基因突变分析

目的　探讨中国人性染色体隐性遗传肾上腺脑白质营养不良 (adrenoleukodystrophy,AL D)的分子发病机理。方法　应用聚合酶链反应结合 DNA测序技术 ,对 4例患者、患者母亲及 2 0名正常对照者的 AL D基因外显子 1、外显子 5及其侧翼进行突变检测。结果　发现 1例患者在 AL D基因外显子 5内含子 5交界处发生了 1875 G→ A突变 ,这种突变可能导致外显子 5和外显子 6剪接异常 ,合成异常蛋白 ,从而使极长链脂肪酸在脑白质、肾上腺及血浆聚集增多 ,导致疾病发生。结论　 AL D基因内含子 5的 5′剪切点突变 ,导致外显子 5和外显子 6剪接异常为性染色体隐性遗传肾上腺脑白质营养不良发病原因之一