X-linked Adrenoleukodystrophy Fibroblasts Research Articles

ABCD1 Transporter Deficiency Results in Altered Cholesterol Homeostasis.

X-linked adrenoleukodystrophy (X-ALD), the most common peroxisomal disorder, is caused by mutations in the peroxisomal transporter ABCD1, resulting in the accumulation of very long-chain fatty acids (VLCFA). Strongly affected cell types, such as oligodendrocytes, adrenocortical cells and macrophages, exhibit high cholesterol turnover. Here, we investigated how ABCD1 deficiency affects cholesterol metabolism in human X-ALD patient-derived fibroblasts and CNS tissues of Abcd1-deficient mice. Lipidome analyses revealed increased levels of cholesterol esters (CE), containing both saturated VLCFA and mono/polyunsaturated (V)LCFA. The elevated CE(26:0) and CE(26:1) levels remained unchanged in LXR agonist-treated Abcd1 KO mice despite reduced total C26:0. Under high-cholesterol loading, gene expression of SOAT1, converting cholesterol to CE and lipid droplet formation were increased in human X-ALD fibroblasts versus healthy control fibroblasts. However, the expression of NCEH1, catalysing CE hydrolysis and the cholesterol transporter ABCA1 and cholesterol efflux were also upregulated. Elevated Soat1 and Abca1 expression and lipid droplet content were confirmed in the spinal cord of X-ALD mice, where expression of the CNS cholesterol transporter Apoe was also elevated. The extent of peroxisome-lipid droplet co-localisation appeared low and was not impaired by ABCD1-deficiency in cholesterol-loaded primary fibroblasts. Finally, addressing steroidogenesis, progesterone-induced cortisol release was amplified in X-ALD fibroblasts. These results link VLCFA to cholesterol homeostasis and justify further consideration of therapeutic approaches towards reducing VLCFA and cholesterol levels in X-ALD.

Antioxidant Response in Human X-Linked Adrenoleukodystrophy Fibroblasts.

Redox imbalance, mitochondrial dysfunction, and inflammation play a major role in the pathophysiology of X-linked adrenoleukodystrophy (X-ALD), an inherited neurodegenerative disease caused by mutations in the ABCD1 gene, encoding the protein responsible for peroxisomal import and degradation of very long chain fatty acids (VLCFAs). Therefore, VLCFAs accumulate in tissues and plasma, constituting a pathognomonic biomarker for diagnosis. However, the precise role of VLCFA accumulation on the diverse clinical phenotypes of X-ALD and the pathogenic link between VLCFAs and oxidative stress remain currently unclear. This study proposes ferroptosis as a crucial contributor to the disease development and progression. The expression profiles of "GPX4-glutathione" and "NQO1-CoQ10" ferroptosis pathways have been analyzed in fibroblasts of one patient with AMN, the late onset and slowly progressive form of X-ALD, and in two patients with cALD, the cerebral inflammatory demyelinating form of early childhood. Furthermore, as no effective treatments are currently available, especially for the rapidly progressing form of X-ALD (cALD), the efficacy of NAC treatment has also been evaluated to open the way toward novel combined therapies. Our findings demonstrate that lipid peroxides accumulate in X-ALD fibroblasts and ferroptosis-counteracting enzymes are dysregulated, highlighting a different antioxidant response in patients with AMN and cALD.

Protective effect of n-acetyl-L-cysteine and rosuvastatin against oxidative stress in fibroblasts from asymptomatic patients with X-ALD: a preliminary study

Introduction: Several studies in the literature have evaluated the role of oxidative stress and adjuvant therapies for X-linked adrenoleukodystrophy (X-ALD). Here, we investigated whether n-acetyl-L-cysteine (NAC) and rosuvastatin (RSV) could influence the generation of reactive species, redox status and nitrative stress in fibroblasts from asymptomatic patients with X-ALD. Methods: Skin biopsy samples were cultured and treated for 2 hours (37 °C) with NAC and RSV. Results: X-ALD fibroblasts generated high levels of reactive oxygen species. These levels were significantly lower in fibroblasts treated with NAC and RSV relative to untreated samples. The X-ALD fibroblasts from asymptomatic patients also had higher catalase activity, and only NAC was able to increase enzyme activity in the samples. Conclusion: Our results indicated that NAC and RSV were able to improve oxidative stress parameters in fibroblasts from asymptomatic patients with X-ALD, showing that adjuvant antioxidant therapy may be a promising treatment strategy for asymptomatic patients with this disease.

Tauroursodeoxycholic bile acid arrests axonal degeneration by inhibiting the unfolded protein response in X-linked adrenoleukodystrophy

The activation of the highly conserved unfolded protein response (UPR) is prominent in the pathogenesis of the most prevalent neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS), which are classically characterized by an accumulation of aggregated or misfolded proteins. This activation is orchestrated by three endoplasmic reticulum (ER) stress sensors: PERK, ATF6 and IRE1. These sensors transduce signals that induce the expression of the UPR gene programme. Here, we first identified an early activator of the UPR and investigated the role of a chronically activated UPR in the pathogenesis of X-linked adrenoleukodystrophy (X-ALD), a neurometabolic disorder that is caused by ABCD1 malfunction; ABCD1 transports very long-chain fatty acids (VLCFA) into peroxisomes. The disease manifests as inflammatory demyelination in the brain or and/or degeneration of corticospinal tracts, thereby resulting in spastic paraplegia, with the accumulation of intracellular VLCFA instead of protein aggregates. Using X-ALD mouse model (Abcd1− and Abcd1−/Abcd2−/− mice) and X-ALD patient’s fibroblasts and brain samples, we discovered an early engagement of the UPR. The response was characterized by the activation of the PERK and ATF6 pathways, but not the IRE1 pathway, showing a difference from the models of AD, PD or ALS. Inhibition of PERK leads to the disruption of homeostasis and increased apoptosis during ER stress induced in X-ALD fibroblasts. Redox imbalance appears to be the mechanism that initiates ER stress in X-ALD. Most importantly, we demonstrated that the bile acid tauroursodeoxycholate (TUDCA) abolishes UPR activation, which results in improvement of axonal degeneration and its associated locomotor impairment in Abcd1−/Abcd2−/− mice. Altogether, our preclinical data provide evidence for establishing the UPR as a key drug target in the pathogenesis cascade. Our study also highlights the potential role of TUDCA as a treatment for X-ALD and other axonopathies in which similar molecular mediators are implicated.

Enzymatic characterization of ELOVL1, a key enzyme in very long-chain fatty acid synthesis

X-linked adrenoleukodystrophy (X-ALD) is a neurometabolic disease that is caused by mutations in the ABCD1 gene. ABCD1 protein deficiency impairs peroxisomal very long-chain fatty acid (VLCFA) degradation resulting in increased cytosolic VLCFA-CoA levels, which are further elongated by the VLCFA-specific elongase, ELOVL1. In adulthood, X-ALD most commonly manifests as a gradually progressive myelopathy (adrenomyeloneuropathy; AMN) without any curative or disease modifying treatments. We recently showed that bezafibrate reduces VLCFA accumulation in X-ALD fibroblasts by inhibiting ELOVL1. Although, in a clinical trial, bezafibrate was unable to lower VLCFA levels in plasma or lymphocytes in X-ALD patients, inhibition of ELOVL1 remains an attractive therapeutic option.In this study, we investigated the kinetic characteristics of ELOVL1 using X-ALD fibroblasts and microsomal fractions from ELOVL1 over-expressing HEK293 cell lines and analyzed the inhibition kinetics of a series of fibrates. Our data show that the CoA esters of bezafibrate and gemfibrozil reduce chain elongation by specifically inhibiting ELOVL1. These fibrates can therefore serve as lead compounds for the development of more potent and more specific inhibitors for ELOVL1.

Abcd2 is a strong modifier of the metabolic impairments in peritoneal macrophages of ABCD1-deficient mice.

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

Structure-Function Analysis of Peroxisomal ATP-binding Cassette Transporters Using Chimeric Dimers

ABCD1 and ABCD2 are two closely related ATP-binding cassette half-transporters predicted to homodimerize and form peroxisomal importers for fatty acyl-CoAs. Available evidence has shown that ABCD1 and ABCD2 display a distinct but overlapping substrate specificity, although much remains to be learned in this respect as well as in their capability to form functional heterodimers. Using a cell model expressing an ABCD2-EGFP fusion protein, we first demonstrated by proximity ligation assay and co-immunoprecipitation assay that ABCD1 interacts with ABCD2. Next, we tested in the pxa1/pxa2Δ yeast mutant the functionality of ABCD1/ABCD2 dimers by expressing chimeric proteins mimicking homo- or heterodimers. For further structure-function analysis of ABCD1/ABCD2 dimers, we expressed chimeric dimers fused to enhanced GFP in human skin fibroblasts of X-linked adrenoleukodystrophy patients. These cells are devoid of ABCD1 and accumulate very long-chain fatty acids (C26:0 and C26:1). We checked that the chimeric proteins were correctly expressed and targeted to the peroxisomes. Very long-chain fatty acid levels were partially restored in transfected X-linked adrenoleukodystrophy fibroblasts regardless of the chimeric construct used, thus demonstrating functionality of both homo- and heterodimers. Interestingly, the level of C24:6 n-3, the immediate precursor of docosahexaenoic acid, was decreased in cells expressing chimeric proteins containing at least one ABCD2 moiety. Our data demonstrate for the first time that both homo- and heterodimers of ABCD1 and ABCD2 are functionally active. Interestingly, the role of ABCD2 (in homo- and heterodimeric forms) in the metabolism of polyunsaturated fatty acids is clearly evidenced, and the chimeric dimers provide a novel tool to study substrate specificity of peroxisomal ATP-binding cassette transporters.

LXR antagonists induce ABCD2 expression

X-linked adrenoleukodystrophy (X-ALD) is a rare neurodegenerative disorder characterized by the accumulation of very-long-chain fatty acids resulting from a β-oxidation defect. Oxidative stress and inflammation are also key components of the pathogenesis. X-ALD is caused by mutations in the ABCD1 gene, which encodes for a peroxisomal half ABC transporter predicted to participate in the entry of VLCFA-CoA into the peroxisome, the unique site of their β-oxidation. Two homologous peroxisomal ABC transporters, ABCD2 and ABCD3 have been proven to compensate for ABCD1 deficiency when overexpressed. Pharmacological induction of these target genes could therefore represent an alternative therapy for X-ALD patients. Since LXR activation was shown to repress ABCD2 expression, we investigated the effects of LXR antagonists in different cell lines. Cells were treated with GSK(17) (a LXR antagonist recently discovered from the GlaxoSmithKline compound collection), 22(S)-hydroxycholesterol (22S-HC, another LXR antagonist) and 22R-HC (an endogenous LXR agonist). We observed up-regulation of ABCD2, ABCD3 and CTNNB1 (the gene encoding for β-catenin, which was recently demonstrated to induce ABCD2 expression) in human HepG2 hepatoma cells and in X-ALD skin fibroblasts treated with LXR antagonists. Interestingly, induction in X-ALD fibroblasts was concomitant with a decrease in oxidative stress. Rats treated with 22S-HC showed hepatic induction of the 3 genes of interest. In human, we show by multiple tissue expression array that expression of ABCD2 appears to be inversely correlated with NR1H3 (LXRα) expression. Altogether, antagonists of LXR that are currently developed in the context of dyslipidemia may find another indication with X-ALD.

Impaired Very Long-chain Acyl-CoA β-Oxidation in Human X-linked Adrenoleukodystrophy Fibroblasts Is a Direct Consequence of ABCD1 Transporter Dysfunction

X-linked adrenoleukodystrophy (X-ALD), an inherited peroxisomal disorder, is caused by mutations in the ABCD1 gene encoding the peroxisomal ATP-binding cassette (ABC) transporter ABCD1 (adrenoleukodystrophy protein, ALDP). Biochemically, X-ALD is characterized by an accumulation of very long-chain fatty acids and partially impaired peroxisomal β-oxidation. In this study, we used primary human fibroblasts from X-ALD and Zellweger syndrome patients to investigate the peroxisomal β-oxidation defect. Our results show that the degradation of C26:0-CoA esters is as severely impaired as degradation of unesterified very long-chain fatty acids in X-ALD and is abolished in Zellweger syndrome. Interestingly, the β-oxidation rates for both C26:0-CoA and C22:0-CoA were similarly affected, although C22:0 does not accumulate in patient fibroblasts. Furthermore, we show that the β-oxidation defect in X-ALD is directly caused by ABCD1 dysfunction as blocking ABCD1 function with a specific antibody reduced β-oxidation to levels observed in X-ALD fibroblasts. By quantification of mRNA and protein levels of the peroxisomal ABC transporters and by blocking with specific antibodies, we found that residual β-oxidation activity toward C26:0-CoA in X-ALD fibroblasts is mediated by ABCD3, although the efficacy of ABCD3 appeared to be much lower than that of ABCD1. Finally, using isolated peroxisomes, we show that β-oxidation of C26:0-CoA is independent of additional CoA but requires a cytosolic factor of >10-kDa molecular mass that is resistant to N-ethylmaleimide and heat inactivation. In conclusion, our findings in human cells suggest that, in contrast to yeast cells, very long-chain acyl-CoA esters are transported into peroxisomes by ABCD1 independently of additional synthetase activity.

ELOVL1 is a potential target for therapeutic intervention in X-linked adrenoleukodystrophy

INTRODUCTION X-linked adrenoleukodystrophy (X-ALD) is a demyelinating disease characterized by high levels of very long-chain fatty acids (VLCFA) in plasma and tissues, due to defi cient transport of VLCFA into peroxisomes, where they would normally be degraded. Prevention or reduction of VLCFA synthesis could represent an interesting therapeutic approach to this disease. This could be achieved through inhibition of ELOVL1, the only elongase catalyzing the elongation of long chain-fatty acids to VLCFA. We have previously shown that VLCFA synthesis and levels were reduced in X-ALD fi broblasts upon treatment with bezafi brate. We have subsequently determined that the CoA ester of bezafi brate inhibits the elongation of C22:0-CoA by ELOVL1. Our research objectives in this study are two-fold: 1) Characterize ELOVL1 and determine the enzyme kinetics; 2) Compare the inhibitory eff ects of the CoA esters of diff erent fi brates on ELOVL1 enzymatic activity.

Caffeic acid phenethyl ester induces adrenoleukodystrophy (Abcd2) gene in human X-ALD fibroblasts and inhibits the proinflammatory response in Abcd1/2 silenced mouse primary astrocytes

X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by mutations in the ABCD1 gene. Accumulation of very long chain fatty acids (VLCFA) that have been attributed to reduced peroxisomal VLCFA β-oxidation activity are the hallmark of the disease. Overexpression of ABCD2 gene, the closest homolog of ABCD1, has been shown to compensate for ABCD1, thus correcting the VLCFA derangement. The accumulation of VLCFA leads to a neuroinflammatory disease process associated with demyelination of the cerebral white matter. The present study underlines the importance of caffeic acid phenethyl ester (CAPE) in inducing the expression of ABCD2 (ALDRP), and normalizing the peroxisomal β-oxidation as well as the levels of saturated and monounsaturated VLCFAs in cultured human skin fibroblasts of X-ALD patients. The expression of ELOVL1, the single elongase catalyzing the synthesis of both saturated VLCFA (C26:0) and mono-unsaturated VLCFA (C26:1), was also reduced by CAPE treatment. Importantly, CAPE upregulated Abcd2 expression and peroxisomal β-oxidation and lowered the VLCFA levels in Abcd1-deficient U87 astrocytes and B12 oligodendrocytes. In addition, using Abcd1/Abcd2-silenced mouse primary astrocytes we examined the effects of CAPE in VLCFA-induced inflammatory response. CAPE treatment decreased the inflammatory response as the expression of inducible nitric oxide synthase, inflammatory cytokine, and activation of NF-κB in Abcd1/Abcd2-silenced mouse primary astrocytes was reduced. The observations indicate that CAPE corrects both the metabolic disease of VLCFA as well as secondary inflammatory disease; therefore, it may be a potential drug candidate to be tested for X-ALD therapy in humans.

Bezafibrate for X-Linked Adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene and is characterized by impaired beta-oxidation of very-long-chain fatty acids (VLCFA) and subsequent VLCFA accumulation in tissues. In adulthood X-ALD most commonly manifests as a gradually progressive myelopathy, (adrenomyeloneuropathy; AMN) without any curative or disease modifying treatments. We recently showed that bezafibrate (BF), a drug used for the treatment of hyperlipidaemia, reduces VLCFA accumulation in X-ALD fibroblasts by inhibiting ELOVL1, an enzyme involved in the VLCFA synthesis. We therefore designed a proof-of-principal clinical trial to determine whether BF reduces VLCFA levels in plasma and lymphocytes of X-ALD patients. Ten males with AMN were treated with BF for 12 weeks at a dose of 400 mg daily, followed by 12 weeks of 800 mg daily. Every 4 weeks patients were evaluated for side effects and blood samples were taken for analysis. Adherence was good as indicated by a clear reduction in triglycerides. There was no reduction in VLCFA in either plasma or lymphocytes. Plasma levels of BF did not exceed 25 µmol/L. We concluded that BF, at least in the dose given, is unable to lower VLCFA levels in plasma or lymphocytes in X-ALD patients. It is unclear whether this is due to the low levels of BF reached in plasma. Our future work is aimed at the identification of highly-specific inhibitors of ELOVL1 that act at much lower concentrations than BF and are well tolerated. BF appears to have no therapeutic utility in X-ALD.Trial RegistrationClinicalTrials.gov NCT01165060

Bezafibrate lowers very long‐chain fatty acids in X‐linked adrenoleukodystrophy fibroblasts by inhibiting fatty acid elongation

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene encoding ALDP, an ATP-binding-cassette (ABC) transporter located in the peroxisomal membrane. ALDP deficiency results in impaired peroxisomal β-oxidation and the subsequent accumulation of very long-chain fatty acids (VLCFA; > C22:0) in plasma and tissues. VLCFA are primarily derived from endogenous synthesis by ELOVL1. Therefore inhibiting this enzyme might constitute a feasible therapeutic approach. In this paper we demonstrate that bezafibrate, a PPAR pan agonist used for the treatment of patients with hyperlipidaemia reduces VLCFA levels in X-ALD fibroblasts. Surprisingly, the VLCFA-lowering effect was independent of PPAR activation and not caused by the increase in either mitochondrial or peroxisomal fatty acid β-oxidation capacity. In fact, our results show that bezafibrate reduces VLCFA synthesis by decreasing the synthesis of C26:0 through a direct inhibition of fatty acid elongation activity. Taken together, our data indicate bezafibrate as a potential pharmacotherapeutic treatment for X-ALD. A clinical trial is currently ongoing to evaluate the effect in patients with X-ALD.Electronic supplementary materialThe online version of this article (doi:10.1007/s10545-012-9471-4) contains supplementary material, which is available to authorized users.

Substrate Specificity Overlap and Interaction between Adrenoleukodystrophy Protein (ALDP/ABCD1) and Adrenoleukodystrophy-related Protein (ALDRP/ABCD2)

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder caused by mutations in the ABCD1 gene, which encodes a peroxisomal member of the ATP-binding cassette (ABC) transporter subfamily D called ALDP. ALDP is supposed to function as a homodimer allowing the entry of CoA-esters of very-long chain fatty acids (VLCFA) into the peroxisome, the unique site of their β-oxidation. ALDP deficiency can be corrected by overexpression of ALDRP, its closest homolog. However, the exact nature of the substrates transported by ALDRP and its relationships with ALDP still remain unclear. To gain insight into the function of ALDRP, we used cell models allowing the induction in a dose-dependent manner of a wild type or a mutated non-functional ALDRP-EGFP fusion protein. We explored the consequences of the changes of ALDRP expression levels on the fatty acid content (saturated, monounsaturated, and polyunsaturated fatty acids) in phospholipids as well as on the levels of β-oxidation of 3 suspected substrates: C26:0, C24:0, and C22:6n-3 (DHA). We found an inverse correlation between the fatty acid content of saturated (C26:0, C24:0) and monounsaturated (C26:1, C24:1) VLCFA and the expression level of ALDRP. Interestingly, we obtained a transdominant-negative effect of the inactive ALDRP-EGFP on ALDP function. This effect is due to a physical interaction between ALDRP and ALDP that we evidenced by proximity ligation assays and coimmunoprecipitation. Finally, the β-oxidation assays demonstrate a role of ALDRP in the metabolism of saturated VLCFA (redundant with that of ALDP) but also a specific involvement of ALDRP in the metabolism of DHA.

The role of ELOVL1 in very long‐chain fatty acid homeostasis and X‐linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene encoding the peroxisomal ABC transporter adrenoleukodystrophy protein (ALDP). X-ALD is characterized by the accumulation of very long-chain fatty acids (VLCFA; ≥C24) in plasma and tissues. In this manuscript we provide insight into the pathway underlying the elevated levels of C26:0 in X-ALD. ALDP transports VLCFacyl-CoA across the peroxisomal membrane. A deficiency in ALDP impairs peroxisomal β-oxidation of VLCFA but also raises cytosolic levels of VLCFacyl-CoA which are substrate for further elongation. We identify ELOVL1 (elongation of very-long-chain-fatty acids) as the single elongase catalysing the synthesis of both saturated VLCFA (C26:0) and mono-unsaturated VLCFA (C26:1). ELOVL1 expression is not increased in X-ALD fibroblasts suggesting that increased levels of C26:0 result from increased substrate availability due to the primary deficiency in ALDP. Importantly, ELOVL1 knockdown reduces elongation of C22:0 to C26:0 and lowers C26:0 levels in X-ALD fibroblasts. Given the likely pathogenic effects of high C26:0 levels, our findings highlight the potential of modulating ELOVL1 activity in the treatment of X-ALD.

Induction of the adrenoleukodystrophy-related gene ( ABCD2) by thyromimetics

X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by mutations in the ABCD1 ( ALD) gene. The ABCD2 gene, its closest homolog, has been shown to compensate for ABCD1 deficiency when overexpressed. We previously demonstrated that the ABCD2 promoter contains a functional thyroid hormone response element. Thyroid hormone (T3) through its receptor TRβ can induce hepatic Abcd2 expression in rodents and transiently normalize the VLCFA level in fibroblasts of Abcd1 null mice. In a therapeutic perspective, the use of selective agonists of TRβ should present the advantage to be devoid of side effects, at least concerning the cardiotoxicity associated to TRα activation. In this study, we compared the effects of T3 with those of two thyromimetics (GC-1 and CGS 23425) specific of TRβ. Using a gene reporter assay, we demonstrated that the rat Abcd2 promoter responds to the thyromimetics in a dose-dependent way similar to what is observed with T3. We then investigated the effects of 2-, 4- and 10-day treatments on the expression of ABCD2 and its paralogs ABCD3 and ABCD4 in human cell lines by RT-qPCR. Both thyromimetics trigger up-regulation of ABCD2–4 genes in HepG2 cells and X-ALD fibroblasts. Interestingly, in X-ALD fibroblasts, while T3 is associated with a transient induction of ABCD2 and ABCD3, the treatments with thyromimetics allow the induction to be maintained until 10 days. Further in vivo experiments in Abcd1 null mice with these thyromimetics should confirm the therapeutic potentialities of these molecules.

Cholesterol-deprivation increases mono-unsaturated very long-chain fatty acids in skin fibroblasts from patients with X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder and is characterized by a striking and unpredictable variation in phenotypic expression. It ranges from a rapidly progressive and fatal cerebral demyelinating disease in childhood (CCALD), to the milder slowly progressive form in adulthood (AMN). 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 reduced beta-oxidation of very long-chain fatty acids (VLCFA, > 22 carbon atoms) in peroxisomes and elevated levels of VLCFA in plasma and tissues. Previously, it has been shown that culturing skin fibroblasts from X-ALD patients in lipoprotein-deficient medium results in reduced VLCFA levels and increased expression of the functionally redundant ALD-related protein (ALDRP). The aim of this study was to further resolve the interaction between cholesterol and VLCFA metabolism in X-ALD. Our data show that the reduction in 26:0 in X-ALD fibroblasts grown in lipoprotein-deficient culture medium (free of cholesterol) is offset by a significant increase in both the level and synthesis of 26:1. We also demonstrate that cholesterol-deprivation results in increased expression of stearoyl-CoA-desaturase (SCD) and increased desaturation of 18:0 to 18:1. Finally, there was no increase in [1- 14C]-26:0 beta-oxidation. Taken together, we conclude that cholesterol-deprivation reduces saturated VLCFA, but increases mono-unsaturated VLCFA. These data may have implications for treatment of X-ALD patients with lovastatin.

Adrenoleukodystrophy: subcellular localization and degradation of adrenoleukodystrophy protein (ALDP/ABCD1) with naturally occurring missense mutations

Mutation in the X-chromosomal adrenoleukodystrophy gene (ALD; ABCD1) leads to X-linked adrenoleukodystrophy (X-ALD), a severe neurodegenerative disorder. The encoded adrenoleukodystrophy protein (ALDP/ABCD1) is a half-size peroxisomal ATP-binding cassette protein of 745 amino acids in humans. In this study, we chose nine arbitrary mutant human ALDP forms (R104C, G116R, Y174C, S342P, Q544R, S606P, S606L, R617H, and H667D) with naturally occurring missense mutations and examined the intracellular behavior. When expressed in X-ALD fibroblasts lacking ALDP, the expression level of mutant His-ALDPs (S606L, R617H, and H667D) was lower than that of wild type and other mutant ALDPs. Furthermore, mutant ALDP-green fluorescence proteins (S606L and H667D) stably expressed in CHO cells were not detected due to rapid degradation. Interestingly, the wild type ALDP co-expressed in these cells also disappeared. In the case of X-ALD fibroblasts from an ALD patient (R617H), the mutant ALDP was not detected in the cells, but appeared upon incubation with a proteasome inhibitor. When CHO cells expressing mutant ALDP-green fluorescence protein (H667D) were cultured in the presence of a proteasome inhibitor, both the mutant and wild type ALDP reappeared. In addition, mutant His-ALDP (Y174C), which has a mutation between transmembrane domain 2 and 3, did not exhibit peroxisomal localization by immunofluorescense study. These results suggest that mutant ALDPs, which have a mutation in the COOH-terminal half of ALDP, including S606L, R617H, and H667D, were degraded by proteasomes after dimerization. Further, the region between transmembrane domain 2 and 3 is important for the targeting of ALDP to the peroxisome.

X-linked adrenoleukodystrophy mice demonstrate abnormalities in cholesterol metabolism

The neurodegenerative disorder X-linked adrenoleukodystrophy (X-ALD) is caused by ABCD1 mutations and characterized by very long-chain fatty acid (VLCFA) accumulation. Cholesterol-lowering normalized VLCFA in fibroblasts and plasma of X-ALD patients. We show that in cultured cells, cholesterol-loading induces ABCD1. In X-ALD mice, plasma cholesterol is elevated and not further increasable by cholesterol-feeding, whereas hepatic HMG-CoA reductase and Abcd2 are downregulated. Upon cholesterol modulation, brain VLCFA increased in X-ALD mice, but decreased in controls. In murine X-ALD fibroblasts, cholesterol-lowering did not normalize VLCFA. Thus, ALDP-deficiency and VLCFA are linked to cholesterol but species differences complicate evaluating cholesterol-lowering drugs 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 β-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 β-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 β-oxidation, and not specific for X-ALD alone. Analysis of plasma samples from patients with X-ALD and different peroxisomal β-oxidation deficiencies revealed increased concentrations of VLCFAs up to 32 carbons. We infer that enhanced elongation does not result from impaired peroxisomal β-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 β-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 β-oxidation disorders.