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Pathophysiology of X-Linked Adrenoleukodystrophy: Updates on Molecular Mechanisms
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Adrenomyeloneuropathy (AMN), the slow progressive phenotype of adrenoleukodystrophy (ALD), has no clinical plasma biomarker for disease progression. This feasibility study aimed to determine whether metabolomics and micro-RNA in blood plasma provide a potential source of biomarkers for AMN disease severity. Metabolomics and RNA-seq were performed on AMN and healthy human blood plasma. Biomarker discovery and pathway analyses were performed using clustering, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and regression against patient's clinical Expanded Disability Status Score (EDSS). Fourteen AMN and six healthy control samples were analyzed. AMN showed strong disease-severity-specific metabolic and miRNA clustering signatures. Strong, significant clinical correlations were shown for 7-alpha-hydroxy-3-oxo-4-cholestenoate (7-HOCA) (r 2 = 0.83, p < 0.00001), dehy-droepiandrosterone sulfate (DHEA-S; r 2 = 0.82, p < 0.00001), hypoxanthine (r 2 = 0.82, p < 0.00001), as well as miRNA-432-5p (r 2 = 0.68, p < 0.00001). KEGG pathway comparison of mild versus severe disease identified affected downstream systems: GAREM, IGF-1, CALCRL, SMAD2&3, glutathione peroxidase, LDH, and NOS. This feasibility study demonstrates that miRNA and metabolomics are a source of potential plasma biomarkers for disease severity in AMN, providing both a disease signature and individual markers with strong clinical correlations. Network analyses of affected systems implicate differentially altered vascular, inflammatory, and oxidative stress pathways, suggesting disease-severity-specific mechanisms as a function of disease severity. K E Y W O R D S adrenoleukodystrophy, adrenomyeloneuropathy, biomarker, leukodystrophy, metabolomics, micro-RNA Bela Rui Turk and Laila Marie Poisson contributed equally to this study.
Aberrant endocannabinoid signaling accompanies several neurodegenerative disorders, including multiple sclerosis. Here, we report altered endocannabinoid signaling in X-linked adrenoleukodystrophy (X-ALD), a rare neurometabolic demyelinating syndrome caused by malfunction of the peroxisomal ABCD1 transporter, resulting in the accumulation of very long-chain fatty acids (VLCFAs). We found abnormal levels of cannabinoid receptor 2 (CB2r) and related endocannabinoid enzymes in the brain and peripheral blood mononuclear cells (PBMCs) of X-ALD patients and in the spinal cord of a murine model of X-ALD. Preclinical treatment with a selective agonist of CB2r (JWH133) halted axonal degeneration and associated locomotor deficits, along with normalization of microgliosis. Moreover, the drug improved the main metabolic disturbances underlying this model, particularly in redox and lipid homeostatic pathways, including increased lipid droplets in motor neurons, through the modulation of the GSK-3β/NRF2 axis. JWH133 inhibited Reactive Oxygen Species elicited by excess VLCFAs in primary microglial cultures of Abcd1-null mice. Furthermore, we uncovered intertwined redox and CB2r signaling in the murine spinal cords and in patient PBMC samples obtained from a phase II clinical trial with antioxidants (NCT01495260). These findings highlight CB2r signaling as a potential therapeutic target for X-ALD and perhaps other neurodegenerative disorders that present with dysregulated redox and lipid homeostasis.
Background: X-linked adrenoleukodystrophy (ALD) is a severe orphan disease caused by mutations in the peroxisomal ABCD1 transporter gene, leading to toxic accumulation of Very Long-Chain Fatty Acids (VLCFA - in particular C26:0) resulting in inflammation, mitochondrial dysfunction and demyelination. AMP-activated protein kinase (AMPK) is downregulated in ALD, and its activation is implicated as a therapeutic target. PXL770 is the first direct allosteric AMPK activator with established clinical efficacy and tolerability. Methods: We investigated its effects in ALD patient-derived fibroblasts/lymphocytes and Abcd1 KO mouse glial cells. Readouts included VLCFA levels, mitochondrial function and mRNA levels of proinflammatory genes and compensatory transporters (ABCD2-3). Following PXL770 treatment in Abcd1 KO mice, we assessed VLCFA levels in tissues, sciatic nerve axonal morphology by electronic microscopy and locomotor function by open-field/balance-beam tests. Results: In patients' cells and Abcd1 KO glial cells, PXL770 substantially decreased C26:0 levels (by ~90%), improved mitochondrial respiration, reduced expression of multiple inflammatory genes and induced expression of ABCD2-3 In Abcd1 KO mice, PXL770 treatment normalized VLCFA in plasma and significantly reduced elevated levels in brain (-25%) and spinal cord (-32%) vs. untreated (p<0.001). Abnormal sciatic nerve axonal morphology was also improved along with amelioration of locomotor function. Conclusion: Direct AMPK activation exerts beneficial effects on several hallmarks of pathology in multiple ALD models in vitro and in vivo, supporting clinical development of PXL770 for this disease. Further studies would be needed to overcome limitations including small sample size for some parameters, lack of additional in vivo biomarkers and incomplete pharmacokinetic characterization. Significance Statement Adrenoleukodystrophy is a rare and debilitating condition with no approved therapies, caused by accumulation of very long-chain fatty acids. AMPK is downregulated in the disease and has been implicated as a potential therapeutic target. PXL770 is a novel clinical stage direct AMPK activator. In these studies, we used PXL770 to achieve preclinical validation of direct AMPK activation for this disease - based on correction of key biochemical and functional readouts in vitro and in vivo, thus supporting clinical development.
X‐linked adrenoleukodystrophy (ALD) results from ABCD1 gene mutations which impair Very Long Chain Fatty Acids (VLCFA; C26:0 and C24:0) peroxisomal import and β‐oxidation, leading to accumulation in plasma and tissues. Excess VLCFA drives impaired cellular functions (e.g. disrupted mitochondrial function), inflammation, and neurodegeneration. Major disease phenotypes include: adrenomyeloneuropathy (AMN), progressive spinal cord axonal degeneration, and cerebral ALD (C‐ALD), inflammatory white matter demyelination and degeneration. No pharmacological treatment is available to‐date for ALD. Pioglitazone, an anti‐diabetic thiazolidinedione, exerts potential benefits in ALD models. Its mechanisms are genomic (PPARγ agonism) and non‐genomic (mitochondrial pyruvate carrier – MPC, long chain acyl‐CoA synthetase 4 – ACSL4, inhibition). However, its use is limited by PPARγ driven side effects (e.g. weight gain, edema). PXL065 is a clinical‐stage deuterium‐stabilized (R)‐enantiomer of pioglitazone which lacks PPARγ agonism but retains MPC activity. Here, we show that incubation of ALD patient‐derived cells (both AMN and C‐ALD) and glial cells from Abcd1‐null mice with PXL065 resulted in: normalization of elevated VLCFA, improved mitochondrial function, and attenuated indices of inflammation. Compensatory peroxisomal transporter gene expression was also induced. Additionally, chronic treatment of Abcd1‐null mice lowered VLCFA in plasma, brain and spinal cord and improved both neural histology (sciatic nerve) and neurobehavioral test performance. Several in vivo effects of PXL065 exceeded those achieved with pioglitazone. PXL065 was confirmed to lack PPARγ agonism but retained ACSL4 activity of pioglitazone. PXL065 has novel actions and mechanisms and exhibits a range of potential benefits in ALD models; further testing of this molecule in ALD patients is warranted. This article is protected by copyright. All rights reserved.
Adrenoleukodystrophy (ALD) is a rare X-linked disease caused by a mutation of the peroxisomal ABCD1 gene. This review summarizes our current understanding of the pathogenic cell- and tissue-specific role of lipid species in the context of experimental therapeutic strategies and provides an overview of critical historical developments, therapeutic trials, and the advent of newborn screening in the United States. In ALD, very long chain fatty acid (VLCFA) chain-length-dependent dysregulation of endoplasmic reticulum stress and mitochondrial radical generating systems inducing cell death pathways has been shown, providing the rationale for therapeutic moiety-specific VLCFA reduction and antioxidant strategies. The continuing increase in newborn screening programs and promising results from ongoing and recent therapeutic investigations provide hope for ALD.
Cerebral adrenoleukodystrophy (cALD) is an inflammatory neurodegenerative disease associated with mutation of the ABCD1 gene. Proteomic analysis of cerebral spinal fluid (CSF) from young males with active cALD revealed markers of inflammation including APOE4. APOE4 genotype has been associated with an inferior prognosis following acute and chronic neurologic injury. We assessed APOE4 inheritance among 83 consecutive young males with cALD prior to hematopoietic cell transplant and its association with markers of cerebral disease. The allele frequency of APOE4 was not significantly different from that of the general population at 17%. Young males with cALD that were APOE4 carriers had similar CSF protein and chitotriosidase activity to that of non-carriers. In contrast, APOE4 carriers had an increased burden of cerebral disease involvement as determined by MRI severity score (10.5 vs 7.0 points, p = 0.01), higher gadolinium intensity score (2.0 vs 1.3 points, p = 0.007), inferior neurologic function (neurologic function score 2.4 vs 1.0, p = 0.001), and elevated CSF MMP2 levels compared to that of non-carriers (13168 vs 9472 pg/mL, p = 0.01). These are the first data showing that APOE4 is associated with increased severity of cerebral disease in cALD and suggest it may be a modifier of disease.
X-linked adrenoleukodystrophy (ALD) is a neurodegenerative peroxisomal disorder with variable clinical phenotypes. Childhood cerebral ALD (CCALD) is at the most severe end of the disease spectrum. In CCALD, the clinical manifestations include increasing deficits in behavior, vision, hearing, coordination, and motor function, as well as seizures. Without treatment, CCALD often results in apparent vegetative state within 1 to 2 years of appearance of initial signs and symptoms. We present the case of a boy with classic inflammatory CCALD who exhibited spontaneous attenuation in disease progression. While extremely rare, spontaneous arrest of disease progression may occur in boys with inflammatory CCALD.
The nuclear factor erythroid 2-like 2 (NRF2) is the master regulator of endogenous antioxidant responses. Oxidative damage is a shared and early-appearing feature in X-linked adrenoleukodystrophy (X-ALD) patients and the mouse model (Abcd1 null mouse). This rare neurometabolic disease is caused by the loss of function of the peroxisomal transporter ABCD1, leading to an accumulation of very long-chain fatty acids and the induction of reactive oxygen species of mitochondrial origin. Here, we identify an impaired NRF2 response caused by aberrant activity of GSK-3β. We find that GSK-3β inhibitors can significantly reactivate the blunted NRF2 response in patients' fibroblasts. In the mouse models (Abcd1- and Abcd1-/Abcd2-/- mice), oral administration of dimethyl fumarate (DMF/BG12/Tecfidera), an NRF2 activator in use for multiple sclerosis, normalized (i) mitochondrial depletion, (ii) bioenergetic failure, (iii) oxidative damage, and (iv) inflammation, highlighting an intricate cross-talk governing energetic and redox homeostasis in X-ALD Importantly, DMF halted axonal degeneration and locomotor disability suggesting that therapies activating NRF2 hold therapeutic potential for X-ALD and other axonopathies with impaired GSK-3β/NRF2 axis.
Epigenomic changes may either cause disease or modulate its expressivity, adding a layer of complexity to mendelian diseases. X‐linked adrenoleukodystrophy (X‐ALD) is a rare neurometabolic condition exhibiting discordant phenotypes, ranging from a childhood cerebral inflammatory demyelination (cALD) to an adult‐onset mild axonopathy in spinal cords (AMN). The AMN form may occur with superimposed inflammatory brain demyelination (cAMN). All patients harbor loss of function mutations in the ABCD1 peroxisomal transporter of very‐long chain fatty acids. The factors that account for the lack of genotype‐phenotype correlation, even within the same family, remain largely unknown.
To gain insight into this matter, here we compared the genome‐wide DNA methylation profiles of morphologically intact frontal white matter areas of children affected by cALD with adult cAMN patients, including male controls in the same age group. We identified a common methylomic signature between the two phenotypes, comprising i) hypermethylation of genes harboring the H3K27me3 mark at promoter regions, ii) hypermethylation of genes with major roles in oligodendrocyte differentiation such as MBP, CNP, MOG and PLP1, and iii) hypomethylation of immune‐associated genes such as IFITM1 and CD59. Moreover, we found increased hypermethylation in CpGs of genes involved in oligodendrocyte differentiation, and also in genes with H3K27me3 marks in their promoter regions in cALD compared with cAMN, correlating with transcriptional and translational changes. Further, using a penalized logistic regression model, we identified the combined methylation levels of SPG20, UNC45A and COL9A3 and also, the combined expression levels of ID4 and MYRF to be good markers capable of discriminating childhood from adult inflammatory phenotypes.
We thus propose the hypothesis that an epigenetically controlled, altered transcriptional program may drive an impaired oligodendrocyte differentiation and aberrant immune activation in X‐ALD patients. These results shed light into disease pathomechanisms and uncover putative biomarkers of interest for prognosis and phenotypic stratification. This article is protected by copyright. All rights reserved.
Background
In X-linked adrenoleukodystrophy, mutations in ABCD1 lead to loss of function of the ALD protein. Cerebral adrenoleukodystrophy is characterized by demyelination and neurodegeneration. Disease progression, which leads to loss of neurologic function and death, can be halted only with allogeneic hematopoietic stem-cell transplantation.
Methods
We enrolled boys with cerebral adrenoleukodystrophy in a single-group, open-label, phase 2–3 safety and efficacy study. Patients were required to have early-stage disease and gadolinium enhancement on magnetic resonance imaging (MRI) at screening. The investigational therapy involved infusion of autologous CD34+ cells transduced with the elivaldogene tavalentivec (Lenti-D) lentiviral vector. In this interim analysis, patients were assessed for the occurrence of graft-versus-host disease, death, and major functional disabilities, as well as changes in neurologic function and in the extent of lesions on MRI. The primary end point was being alive and having no major functional disability at 24 months after infusion.
Results
A total of 17 boys received Lenti-D gene therapy. At the time of the interim analysis, the median follow-up was 29.4 months (range, 21.6 to 42.0). All the patients had gene-marked cells after engraftment, with no evidence of preferential integration near known oncogenes or clonal outgrowth. Measurable ALD protein was observed in all the patients. No treatment-related death or graft-versus-host disease had been reported; 15 of the 17 patients (88%) were alive and free of major functional disability, with minimal clinical symptoms. One patient, who had had rapid neurologic deterioration, had died from disease progression. Another patient, who had had evidence of disease progression on MRI, had withdrawn from the study to undergo allogeneic stem-cell transplantation and later died from transplantation-related complications.
Conclusions
Early results of this study suggest that Lenti-D gene therapy may be a safe and effective alternative to allogeneic stem-cell transplantation in boys with early-stage cerebral adrenoleukodystrophy. Additional follow-up is needed to fully assess the duration of response and long-term safety. (Funded by Bluebird Bio and others; STARBEAM ClinicalTrials.gov number, NCT01896102; ClinicalTrialsRegister.eu number, 2011-001953-10.)
Activation of Cerebral X-linked Adrenoleukodystrophy After Head Trauma - Volume 44 Issue 5 - Adrian Budhram, Sachin K. Pandey
X-linked adrenoleukodystrophy (ALD) is a severe neurodegenerative disorder caused by the accumulation of very long-chain fatty acids (VLCFA) due to mutations in the ABCD1 gene. The phenotypic spectrum ranges from a fatal cerebral demyelinating disease in childhood (cerebral ALD) to a progressive myelopathy without cerebral involvement in adulthood (adrenomyeloneuropathy). Because ABCD1 mutations have no predictive value with respect to clinical outcome a role for modifier genes was postulated. We report that the CYP4F2 polymorphism rs2108622 increases the risk of developing cerebral ALD in Caucasian patients. The rs2108622 polymorphism (c.1297G>A) results in an amino acid substitution valine for methionine at position 433 (p.V433M). Using cellular models of VLCFA accumulation, we show that p.V433M decreases the conversion of VLCFA into very long-chain dicarboxylic acids by ω-oxidation, a potential escape route for the deficient peroxisomal β-oxidation of VLCFA in ALD. Although p.V433M does not affect the catalytic activity of CYP4F2 it reduces CYP4F2 protein levels markedly. These findings open perspectives for therapeutic interventions in a disease with currently limited treatment options.
X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by mutations in the ABCD1 gene, leading to a defect in the peroxisomal adrenoleukodystrophy protein (ALDP), which inhibits the β-oxidation of very long chain fatty acids (VLCFAs). It is a complex disease where the same mutation in the peroxisomal ABCD1 can lead to clinically diverse phenotypes ranging from the fatal disorder of cerebral ALD (cALD) to mild adult disorder of adrenomyeloneuropathy (AMN). This suggests a role of epigenetic factors/modifier genes in disease progression of X-ALD which is not understood at present. To examine the possible role of microRNA (miRNA) in X-ALD disease mechanisms for differences in cALD and AMN phenotype, we profiled 1008 known miRNA in cALD, AMN, and normal human skin fibroblasts using miScript miRNA PCR array (Qiagen) and selected miRNAs which had differential expression in cALD and AMN fibroblasts. Eleven miRNA which were differentially regulated in cALD and AMN fibroblasts were identified. miR-196a showed a significant differential expression between cALD and AMN and is further characterized for target gene regulation. The predicted role of miR-196a in inhibition of inflammatory signaling factors (IKKα and IKKβ) and ELOVL1 expression suggests the pathological role of altered expression of miR-196a. This study indicates that miR-196a participated in differential regulation of ELOVL1 and inflammatory response between cALD as compared to AMN and may be a possible biomarker to differentiate between cALD and AMN.
X-linked adrenoleukodystrophy (X-ALD) is caused by mutations and/or deletions in the ABCD1 gene. Similar mutations/deletions can give rise to variable phenotypes ranging from mild adrenomyeloneuropathy (AMN) to inflammatory fatal cerebral adrenoleukodystrophy (ALD) via unknown mechanisms. We recently reported the loss of the anti-inflammatory protein adenosine monophosphate activated protein kinase (AMPK alpha 1) exclusively in ALD patient-derived cells. X-ALD mouse model (Abcd1-knockout (KO) mice) mimics the human AMN phenotype and does not develop the cerebral inflammation characteristic of human ALD. In this study we document that AMPK alpha 1 levels in vivo (in brain cortex and spinal cord) and in vitro in Abcd1-KO mixed glial cells are similar to that of wild type mice. Deletion of AMPK alpha 1 in the mixed glial cells of Abcd1-KO mice induced spontaneous mitochondrial dysfunction (lower oxygen consumption rate and ATP levels). Mitochondrial dysfunction in ALD patient-derived cells and in AMPK alpha 1-deleted Abcd1-KO mice mixed glial cells was accompanied by lower levels of mitochondrial complex (1-V) subunits. More importantly, AMPK alpha 1 deletion induced proinflammatory inducible nitric oxide synthase levels in the unstimulated Abcd1-KO mice mixed glial cells. Taken together, this study provides novel direct evidence for a causal role for AMPK loss in the development of mitochondrial dysfunction and proinflammatory response in X-ALD.
Metformin is a first-line antidiabetic agent taken by 150 million people across the world every year, yet its mechanism remains
only partially understood and controversial. It was proposed that suppression of glucose production in hepatocytes by metformin
is AMPK-independent; however, unachievably high concentrations of metformin were employed in these studies. In the current
study, we find that metformin, via an AMP-activated protein kinase (AMPK)-dependent mechanism, suppresses glucose production
and gluconeogenic gene expression in primary hepatocytes at concentrations found in the portal vein of animals (60–80 μm). Metformin also inhibits gluconeogenic gene expression in the liver of mice administered orally with metformin. Furthermore,
the cAMP-PKA pathway negatively regulates AMPK activity through phosphorylation at Ser-485/497 on the α subunit, which in
turn reduces net phosphorylation at Thr-172. Because diabetic patients often have hyperglucagonemia, AMPKα phosphorylation
at Ser-485/497 is a therapeutic target to improve metformin efficacy.
Fatty acids (FAs) are highly diverse in terms of carbon (C) chain-length and number of double bonds. FAs with C>20 are called very long-chain fatty acids (VLCFAs). VLCFAs are found not only as constituents of cellular lipids such as sphingolipids and glycerophospholipids but also as precursors of lipid mediators. Our understanding on the function of VLCFAs is growing in parallel with the identification of enzymes involved in VLCFA synthesis or degradation. A variety of inherited diseases, such as ichthyosis, macular degeneration, myopathy, mental retardation, and demyelination, are caused by mutations in the genes encoding VLCFA metabolizing enzymes. In this review, we describe mammalian VLCFAs by highlighting their tissue distribution and metabolic pathways, and we discuss responsible genes and enzymes with reference to their roles in pathophysiology.
Currently the molecular basis for the clinical heterogeneity of X-linked adrenoleukodystrophy (X-ALD) is poorly understood. The genetic bases for all different phenotypic variants of X-ALD are mutations in the gene encoding the peroxisomal ATP-binding cassette (ABC) transporter, ABCD1 (formerly adrenoleukodystrophy protein, ALDP). ABCD1 transports CoA-activated very long-chain fatty acids from the cytosol into the peroxisome for degradation. The phenotypic variability is remarkable ranging from cerebral inflammatory demyelination of childhood onset, leading to death within a few years, to adults remaining presymptomatic through more than five decades. There is no general genotype-phenotype correlation in X-ALD. The default manifestation of mutations in ABCD1 is adrenomyeloneuropathy, a slowly progressive dying-back axonopathy affecting both ascending and descending spinal cord tracts as well as in some cases, a peripheral neuropathy. In about 60% of male X-ALD patients, either in childhood (35-40%) or in adulthood (20%), an initial, clinically silent, myelin destabilization results in conversion to a devastating, rapidly progressive form of cerebral inflammatory demyelination. Here, ABCD1 remains a susceptibility gene, necessary but not sufficient for inflammatory demyelination to occur. Although the accumulation of very long-chain fatty acids appears to be essential for the pathomechanism of all phenotypes, the molecular mechanisms underlying these phenotypes are fundamentally different. Cell autonomous processes such as oxidative stress and energy shortage in axons as well as non-cell autonomous processes involving axon-glial interactions seem pertinent to the dying-back axonopathy. Various dynamic mechanisms may underlie the initiation of inflammation, the altered immune reactivity, the propagation of inflammation, as well as the mechanisms leading to the arrest of inflammation after hematopoietic stem cell transplantation. An improved understanding of the molecular mechanisms involved in these events is required for the development of urgently needed therapeutics.
Background
X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder of X-linked inheritance caused by a mutation in the ABCD1 gene which determines an accumulation of long-chain fatty acids in plasma and tissues. Recent evidence shows that oxidative stress may be a hallmark in the pathogenesis of X-ALD and glutathione plays an important role in the defense against free radicals. In this study we have analyzed glutathione homeostasis in lymphocytes of 14 patients with X-ALD and evaluated the balance between oxidized and reduced forms of glutathione, in order to define the role of this crucial redox marker in this condition.
Methods
Lymphocytes, plasma and erythrocytes were obtained from the whole blood of 14 subjects with X-ALD and in 30 healthy subjects. Total, reduced and protein-bound glutathione levels were measured in lymphocytes by HPLC analysis. Erythrocyte free glutathione and antioxidant enzyme activities, plasma thiols and carbonyl content were determined by spectrophotometric assays.
Results
A significant decrease of total and reduced glutathione was found in lymphocytes of patients, associated to high levels of all oxidized glutathione forms. A decline of free glutathione was particularly significant in erythrocytes. The increased oxidative stress in X-ALD was additionally confirmed by the decrease of plasma thiols and the high level of carbonyls.
Conclusion
Our results strongly support a role for oxidative stress in the pathophysiology of X-ALD and strengthen the importance of the balance among glutathione forms as a hallmark and a potential biomarker of the disease.
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.
X-linked adrenoleukodystrophy (X-ALD) is an inherited metabolic disorder of the nervous system characterized by axonopathy in spinal cords and/or cerebral demyelination, adrenal insufficiency and accumulation of very long-chain fatty acids (VLCFA) in plasma and tissues. The disease is caused by malfunction of the ABCD1 gene, which encodes a peroxisomal transporter of VLCFA or VLCFA-CoA. In the mouse, ABCD1 loss causes late onset axonal degeneration in the spinal cord, associated with locomotor disability resembling the most common phenotype in patients, adrenomyeloneuropathy. We have formerly shown that an excess of the VLCFA C26:0 induces oxidative damage, which underlies the axonal degeneration exhibited by the Abcd1(-) mice. In the present study, we sought to investigate the noxious effects of C26:0 on mitochondria function. Our data indicate that in X-ALD patients' fibroblasts, excess of C26:0 generates mtDNA oxidation and specifically impairs oxidative phosphorylation triggering mitochondrial ROS production from electron transport chain complexes. This correlates with impaired Complex V phosphorylative activity, as visualized by high-resolution respirometry on spinal cord slices of Abcd1(-) mice. Further, we identified a marked oxidation of key OXPHOS system subunits in Abcd1(-) mouse spinal cords at presymptomatic stages. Altogether, our results illustrate some of the mechanistic intricacies by which the excess of a fatty acid targeted to peroxisomes, activates a deleterious process of oxidative damage to mitochondria, leading to a multifaceted dysfunction of this organelle. These findings may be of relevance for patient management while unveiling novel therapeutic targets for X-ALD.
X-linked adrenoleukodystrophy (X-ALD) is an inherited neurodegenerative disorder caused by mutations in the gene. Approximately 20% of X-ALD female carriers may develop neurological symptoms. Skewed X chromosome inactivation (XCI) has been proposed to influence the manifestation of symptoms in X-ALD carriers, but data remain conflicting so far. We identified a three generation kindred, with five heterozygous females, including two manifesting carriers. XCI pattern and the allele expression were assessed in order to determine if symptoms in X-ALD carriers could be related to skewed XCI and whether skewing within this family is more consistent with genetically influenced or completely random XCI.
We found a high frequency of skewing in this family. Four of five females had skewed XCI, including two manifesting carriers favoring the mutant allele, one asymptomatic carrier favoring the normal allele, and one female who was not an X-ALD carrier. Known causes of skewing, such as chromosomal abnormalities, selection against deleterious alleles, promoter mutations, were not consistent with our results.
Our data support that skewed XCI in favor of the mutant allele would be associated with the manifestation of heterozygous symptoms. Furthermore, XCI skewing in this family is genetically influenced. However, the underlying mechanism remains to be substantiated by further experiments.
X-linked adrenoleukodystrophy (X-ALD) is the most frequent peroxisomal disease. The two main clinical phenotypes of X-ALD are adrenomyeloneuropathy (AMN) and inflammatory cerebral ALD that manifests either in children or more rarely in adults. About 65% of heterozygote females develop symptoms by the age of 60years. Mutations in the ABCD1 gene affect the function of the encoded protein ALDP, an ATP-binding-cassette (ABC) transporter located in the peroxisomal membrane protein. ALDP deficiency impairs the peroxisomal beta-oxidation of very long-chain fatty acids (VLCFA) and facilitates their further chain elongation by ELOVL1 resulting in accumulation of VLCFA in plasma and tissues. While all patients have mutations in the ABCD1 gene, there is no general genotype-phenotype correlation. Environmental factors and a multitude of modifying genes appear to determine the clinical manifestation in this monogenetic but multifactorial disease. This review focuses on the clinical, biochemical, genetic and pathophysiological aspects of X-ALD. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease.
X-linked adrenoleukodystrophy (ALD) is a metabolic, peroxisomal disease that results from a mutation in the ABCD1 gene. The most severe course of ALD progression is the cerebral inflammatory and demyelinating form of the disease, cALD. To date there is very little information on the cytokine mediators in the cerebral spinal fluid (CSF) of these boys.
Measurement of 23 different cytokines was performed on CSF and serum of boys with cerebral ALD and patients without ALD. Significant elevations in CSF IL-8 (29.3±2.2 vs 12.8±1.1 pg/ml, p = 0.0001), IL-1ra (166±30 vs 8.6±6.5 pg/ml, p = 0.005), MCP-1 (610±47 vs 328±34 pg/ml, p = 0.002), and MIP-1b (14.2±1.3 vs 2.0±1.4 pg/ml, p<0.0001) were found in boys with cALD versus the control group. The only serum cytokine showing an elevation in the ALD group was SDF-1 (2124±155 vs 1175±125 pg/ml, p = 0.0001). The CSF cytokines of IL-8 and MCP-1b correlated with the Loes MRI severity score (p = 0.04 and p = 0.008 respectively), as well as the serum SDF-1 level (p = 0.002). Finally, CSF total protein was also significantly elevated in boys with cALD and correlated with both IL-8, MCP-1b (p = 0.0001 for both), as well as Loes MRI severity score (p = 0.0007).
IL-8, IL-1ra, MCP-1, MIP-1b and CSF total protein were significantly elevated in patients with cALD; IL-8, MCP-1b, and CSF total protein levels correlated with disease severity determined by MRI. This is the largest report of CSF cytokine levels in cALD to date, and identification of these key cytokines will provide further insight into disease progression and perhaps lead to improved targeted therapies.
X-linked adrenoleukodystrophy (X-ALD) is an inherited neurodegenerative disorder expressed as four disease variants characterized by adrenal insufficiency and graded damage in the nervous system. X-ALD is caused by a loss of function of the peroxisomal ABCD1 fatty-acid transporter, resulting in the accumulation of very long chain fatty acids (VLCFA) in the organs and plasma, which have potentially toxic effects in CNS and adrenal glands. We have recently shown that treatment with a combination of antioxidants containing α-tocopherol, N-acetyl-cysteine and α-lipoic acid reversed oxidative damage and energetic failure, together with the axonal degeneration and locomotor impairment displayed by Abcd1 null mice, the animal model of X-ALD. This is the first direct demonstration that oxidative stress, which is a hallmark not only of X-ALD, but also of other neurodegenerative processes, such as Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD), contributes to axonal damage. The purpose of this review is, first, to discuss the molecular and cellular underpinnings of VLCFA-induced oxidative stress, and how it interacts with energy metabolism and/or inflammation to generate a complex syndrome wherein multiple factors are contributing. Particular attention will be paid to the dysregulation of redox homeostasis by the interplay between peroxisomes and mitochondria. Second, we will extend this analysis to the aforementioned neurodegenerative diseases with the aim of defining differences as well as the existence of a core pathogenic mechanism that would justify the exchange of therapeutic opportunities among these pathologies. This article is part of a Special Issue entitled: Metabolic functions and biogenesis of peroxisomes in health and disease.
Approximately 20% of adrenoleukodystrophy (X-ALD) female carriers may develop clinical manifestations, typically consisting of progressive spastic gait, sensory deficits and bladder dysfunctions. A skewing in X Chromosome Inactivation (XCI), leading to the preferential expression of the X chromosome carrying the mutant ABCD1 allele, has been proposed as a mechanism influencing X-linked adrenoleukodystrophy (X-ALD) carrier phenotype, but reported data so far are conflicting.
To shed light into this topic we assessed the XCI pattern in peripheral blood mononuclear cells (PBMCs) of 30 X-ALD carriers. Since a frequent problem with XCI studies is the underestimation of skewing due to an incomplete sample digestion by restriction enzymes, leading to variable results, we developed a pyrosequencing assay to identify samples completely digested, on which to perform the XCI assay. Pyrosequencing was also used to quantify ABCD1 allele-specific expression. Moreover, very long-chain fatty acid (VLCFA) levels were determined in the same patients.
We found severely (≥90:10) or moderately (≥75:25) skewed XCI in 23 out of 30 (77%) X-ALD carriers and proved that preferential XCI is mainly associated with the preferential expression of the mutant ABCD1 allele, irrespective of the manifestation of symptoms. The expression of mutant ABCD1 allele also correlates with plasma VLCFA concentrations.
Our results indicate that preferential XCI leads to the favored expression of the mutant ABCD1 allele. This emerges as a general phenomenon in X-ALD carriers not related to the presence of symptoms. Our data support the postulated growth advantage of cells with the preferential expression of the mutant ABCD1 allele, but argue against the use of XCI pattern, ABCD1 allele-specific expression pattern and VLCFA plasma concentration as biomarkers to predict the development of symptoms in X-ALD carriers.
X-linked adrenoleukodystrophy (XALD), a neurological disorder caused by mutations in the peroxisomal membrane protein gene ABCD1, presents as a rapidly progressing, inflammatory cerebral demyelination (cerebral cases) or a slowly progressing, distal axonopathy (non-cerebral cases). Specific ABCD1 defects do not explain this significant phenotypic variation. Patients have increased plasma and tissue very long chain fatty acid levels and increased cellular oxidative stress and oxidative damage. Superoxide dismutase 2 (SOD2), at candidate modifier locus 6q25.3, detoxifies superoxide radicals protecting against oxidative stress and damage. We tested an SOD2 variant C47T (Ala16Val) associated with reduced enzymatic activity as a potential modifier gene of cerebral demyelinating disease by comparing 117 cerebral XALD cases with 105 non-cerebral XALD cases. The hypoactive valine allele of the variant was associated with cerebral disease under a dominant model in the full data set (p = 0.04; ORT* = 1.90, 95% CI 1.01-3.56) and the non-childhood cerebral disease subset (p = 0.03; ORT* = 2.47, 95% CI 1.08-5.61). Three tag SNPs were genotyped to test for additional SNP or haplotype associations. A common haplotype, GTAC, which included the SOD2 valine allele, was associated with cerebral disease in the full data set (p = 0.03; OR = 1.75, 95% CI 1.11-2.75) and the non-childhood cerebral disease subset (p = 0.008; OR = 2.20, 95% CI 1.27-3.83). There was no association between childhood cerebral XALD and the C47T variant or the GTAC haplotype. Thus, reduced SOD2 activity may contribute to the development of cerebral demyelination in adolescent and adult XALD patients.
Individuals who are obese are frequently insulin resistant, putting them at increased risk of developing type 2 diabetes and its associated adverse health conditions. The accumulation in adipose tissue of macrophages in an inflammatory state is a hallmark of obesity-induced insulin resistance. Here, we reveal a role for AMPK β1 in protecting macrophages from inflammation under high lipid exposure. Genetic deletion of the AMPK β1 subunit in mice (referred to herein as β1(-/-) mice) reduced macrophage AMPK activity, acetyl-CoA carboxylase phosphorylation, and mitochondrial content, resulting in reduced rates of fatty acid oxidation. β1(-/-) macrophages displayed increased levels of diacylglycerol and markers of inflammation, effects that were reproduced in WT macrophages by inhibiting fatty acid oxidation and, conversely, prevented by pharmacological activation of AMPK β1-containing complexes. The effect of AMPK β1 loss in macrophages was tested in vivo by transplantation of bone marrow from WT or β1(-/-) mice into WT recipients. When challenged with a high-fat diet, mice that received β1(-/-) bone marrow displayed enhanced adipose tissue macrophage inflammation and liver insulin resistance compared with animals that received WT bone marrow. Thus, activation of AMPK β1 and increasing fatty acid oxidation in macrophages may represent a new therapeutic approach for the treatment of insulin resistance.
Cerebral adrenoleukodystrophy (cALD) remains a devastating neurodegenerative disease; only allogeneic hematopoietic cell transplantation (HCT) has been shown to provide long-term disease stabilization and survival. Sixty boys undergoing HCT for cALD from 2000 to 2009 were analyzed. The median age at HCT was 8.7 years; conditioning regimens and allograft sources varied. At HCT, 50% demonstrated a Loes radiographic severity score ≥ 10, and 62% showed clinical evidence of neurologic dysfunction. A total of 78% (n = 47) are alive at a median 3.7 years after HCT. The estimate of 5-year survival for boys with Loes score < 10 at HCT was 89%, whereas that for boys with Loes score ≥ 10 was 60% (P = .03). The 5-year survival estimate for boys absent of clinical cerebral disease at HCT was 91%, whereas that for boys with neurologic dysfunction was 66% (P = .08). The cumulative incidence of transplantation-related mortality at day 100 was 8%. Post-transplantation progression of neurologic dysfunction depended significantly on the pre-HCT Loes score and clinical neurologic status. We describe the largest single-institution analysis of survival and neurologic function outcomes after HCT in cALD. These trials were registered at www.clinicaltrials.gov as #NCT00176904, #NCT00668564, and #NCT00383448.
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; > or =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 beta-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.
X-linked adrenoleukodystrophy (ALD) is a severe brain demyelinating disease in boys that is caused by a deficiency in ALD
protein, an adenosine triphosphate–binding cassette transporter encoded by the ABCD1 gene. ALD progression can be halted by allogeneic hematopoietic cell transplantation (HCT). We initiated a gene therapy trial
in two ALD patients for whom there were no matched donors. Autologous CD34+ cells were removed from the patients, genetically corrected ex vivo with a lentiviral vector encoding wild-type ABCD1, and then re-infused into the patients after they had received myeloablative treatment. Over a span of 24 to 30 months of
follow-up, we detected polyclonal reconstitution, with 9 to 14% of granulocytes, monocytes, and T and B lymphocytes expressing
the ALD protein. These results strongly suggest that hematopoietic stem cells were transduced in the patients. Beginning 14
to 16 months after infusion of the genetically corrected cells, progressive cerebral demyelination in the two patients stopped,
a clinical outcome comparable to that achieved by allogeneic HCT. Thus, lentiviral-mediated gene therapy of hematopoietic
stem cells can provide clinical benefits in ALD.
X-linked adrenoleukodystrophy is a severe demyelinating neurodegenerative disease mainly affecting males. The severe cerebral adrenoleukodystrophy (cALD) phenotype has a poor prognosis and underlying mechanism of onset and progression of neuropathology remains poorly understood. In this study we aim to integrate metabolomic and microRNA (miRNA) datasets to identify variances associated with cALD. Postmortem brain tissue samples from five healthy controls (CTL) and five cALD patients were utilized in this study. White matter from ALD patients was obtained from normal-appearing areas, away from lesions (NLA) and from the periphery of lesions- plaque shadow (PLS). Metabolomics was performed by gas chromatography coupled with time-of-flight mass spectrometry and miRNA expression analysis was performed by next generation sequencing (RNAseq). Principal component analysis revealed that among the three sample groups (CTL, NLA and PLS) there were 19 miRNA, including several novel miRNA, of which 17 were increased with disease severity and 2 were decreased. Untargeted metabolomics revealed 13 metabolites with disease severity-related patterns with 7 increased and 6 decreased with disease severity. Ingenuity pathway analysis of differentially altered metabolites and miRNA comparing CTL with NLA and NLA with PLS, identified several hubs of metabolite and signaling molecules and their upstream regulation by miRNA. The transomic approach to map the crosstalk between miRNA and metabolomics suggests involvement of specific molecular and metabolic pathways in cALD and offers opportunity to understand the complex underlying mechanism of disease severity in cALD.
X‐linked adrenoleukodystrophy (X‐ ALD ) is a progressive neurometabolic disease caused by mutations/deletions in the Abcd1 gene. Similar mutations/deletions in the Abcd1 gene often result in diagonally opposing phenotypes of mild adrenomyeloneuropathy and severe neuroinflammatory cerebral adrenoleukodystrophy ( ALD ), which suggests involvement of downstream modifier genes. We recently documented the first evidence of loss of AMP ‐activated protein kinase α1 ( AMPK α1) in ALD patient‐derived cells. Here, we report the novel loss of AMPK α1 in postmortem brain white matter of patients with ALD phenotype. Pharmacological activation of AMPK can rescue the mitochondrial dysfunction and inhibit the pro‐inflammatory response. The FDA approved anti‐diabetic drug Metformin, a well‐known AMPK activator, induces mitochondrial biogenesis and is documented for its anti‐inflammatory role. We observed a dose‐dependent activation of AMPK α1 in metformin‐treated X‐ ALD patient‐derived fibroblasts. Metformin also induced mitochondrial oxidative phosphorylation and ATP levels in X‐ ALD patient‐derived fibroblasts. Metformin treatment decreased very long chain fatty acid levels and pro‐inflammatory cytokine gene expressions in X‐ ALD patient‐derived cells. Abcd2 [adrenoleukodystrophy protein‐related protein] levels were increased in metformin‐treated X‐ ALD patient‐derived fibroblasts and Abcd1‐ KO mice primary mixed glial cells. Abcd2 induction was AMPK α1‐dependent since metformin failed to induce Abcd2 levels in AMPK α1‐ KO mice‐derived primary mixed glial cells. In vivo metformin (100 mg/Kg) in drinking water for 60 days induced Abcd2 levels and mitochondrial oxidative phosphorylation protein levels in the brain and spinal cord of Abcd1‐ KO mice. Taken together, these results provide proof‐of‐principle for therapeutic potential of metformin as a useful strategy for correcting the metabolic and inflammatory derangements in X‐ ALD by targeting AMPK .
image There is no effective therapy for inherited peroxisomal disorder X‐linked adrenoleukodystrophy (X‐ ALD ). We document the therapeutic potential of FDA approved drug, Metformin, for X‐ ALD by targeting AMPK . Metformin induced peroxisomal Abcd2 levels in vitro and in vivo . Metformin lowered VLCFA levels, improved mitochondrial function and ameliorated inflammatory gene expression in X‐ ALD patient‐derived cells. Metformin‐induced Abcd2 levels were dependent on AMPK α1, a metabolic and anti‐inflammatory gene, recently documented by our laboratory to play a putative role in X‐ ALD pathology.
Read the Editorial Highlight for this article on page 10 .
The underlying mechanism(s) for development of the inflammatory response in inherited, fatal neurometabolic disease X-linked adrenoleukodystrophy (X-ALD) remain completely unknown. Genetic defect (ABCD1 mutation/ deletion), common to all phenotypes of X-ALD, has failed to explain the development of inflammation only in a subset of patients. In this study we document the novel role of microRNAs (miRNAs) in the development of the inflammatory response in unstimulated ALD patient-derived lymphocytes and Abcd1-knockout (Abcd1-KO) mice mixed glial cells. The levels of proinflammatory cytokine gene expression (inducible nitric oxide synthase [iNOS]) were increased in X-ALD patient-derived lymphocytes. Predictions via the use of online bioinformatics algorithms and confirmed by using miRNA mimic of inhibitor-transfection method (gain- and loss-of-function) revealed the role of miR-323-5p in regulating iNOS expression in X-ALD patient-derived lymphocytes. Functional confirmation of the targets was obtained by using the dual-luciferase assay and western blot analysis. Abcd1-KO mice do not develop the inflammatory response characteristic of the fatal X-ALD phenotype. We recently reported that AMP-activated protein kinase (AMPKα1) deletion induced spontaneous iNOS expression in Abcd1-KO mice mixed glial cells. Here we discover the novel role of miR-323-5p regulating the iNOS response in AMPKα1-deleted Abcd1-KO mice mixed glial cells. This study demonstrated the novel role of miR-323-5p in regulating the inflammatory response in unstimulated X-ALD patient-derived cells and mixed glial cells from Abcd1-KO mice suggesting that these miRNA could function as promising novel therapeutic targets for the treatment of X-ALD.
To describe a diagnostic protocol, surveillance and treatment guidelines, genetic counseling considerations and long-term follow-up data elements developed in preparation for X-linked adrenoleukodystrophy (X-ALD) newborn screening in New York State.
A group including the director from each regional NYS inherited metabolic disorder center, personnel from the NYS Newborn Screening Program, and others prepared a follow-up plan for X-ALD NBS. Over the months preceding the start of screening, a series of conference calls took place to develop and refine a complete newborn screening system from initial positive screen results to long-term follow-up.
A diagnostic protocol was developed to determine for each newborn with a positive screen whether the final diagnosis is X-ALD, carrier of X-ALD, Zellweger spectrum disorder, acyl CoA oxidase deficiency or D-bifunctional protein deficiency. For asymptomatic males with X-ALD, surveillance protocols were developed for use at the time of diagnosis, during childhood and during adulthood. Considerations for timing of treatment of adrenal and cerebral disease were developed.
Because New York was the first newborn screening laboratory to include X-ALD on its panel, and symptoms may not develop for years, long-term follow-up is needed to evaluate the presented guidelines.
Copyright © 2015 Elsevier Inc. All rights reserved.
Macrophages control the resolution of inflammation through the transition from a proinflammatory (M1) to an anti-inflammatory (M2) phenotype. Here, we present evidence for a role of AMPKα1, a master regulator of energy homeostasis, in macrophage skewing that occurs during skeletal muscle regeneration. Muscle regeneration was impaired in AMPKα1(-/-) mice. In vivo loss-of-function (LysM-Cre;AMPKα1(fl/fl) mouse) and rescue (bone marrow transplantation) experiments showed that macrophagic AMPKα1 was required for muscle regeneration. Cell-based experiments revealed that AMPKα1(-/-) macrophages did not fully acquire the phenotype or the functions of M2 cells. In vivo, AMPKα1(-/-) leukocytes did not acquire the expression of M2 markers during muscle regeneration. Skewing from M1 toward M2 phenotype upon phagocytosis of necrotic and apoptotic cells was impaired in AMPKα1(-/-) macrophages and when AMPK activation was prevented by the inhibition of its upstream activator, CaMKKβ. In conclusion, AMPKα1 is crucial for phagocytosis-induced macrophage skewing from a pro- to anti-inflammatory phenotype at the time of resolution of inflammation.
AMPK is a serine/threonine kinase that regulates energy homeostasis and metabolic stress in eukaryotes. Previous work from our laboratory, as well as by others, has provided evidence that AMPKα1 acts as a negative regulator of TLR-induced inflammatory function. Herein, we demonstrate that AMPKα1-deficient macrophages and DCs exhibit heightened inflammatory function and an enhanced capacity for antigen presentation favoring the promotion of Th1 and Th17 responses. Macrophages and DCs generated from AMPKα1-deficient mice produced higher levels of proinflammatory cytokines and decreased production of the anti-inflammatory cytokine IL-10 in response to TLR and CD40 stimulation as compared with WT cells. In assays of antigen presentation, AMPKα1 deficiency in the myeloid APC and T cell populations contributed to enhanced IL-17 and IFN-γ production. Focusing on the CD154-CD40 interaction, we found that CD40 stimulation resulted in increased phosphorylation of ERK1/2, p38, and NF-κB p65 and decreased activation of the anti-inflammatory Akt -GSK3β-CREB pathway in DCs deficient for AMPKα1. Our data demonstrate that AMPKα1 serves to attenuate LPS and CD40-mediated proinflammatory activity of myeloid APCs and that AMPKα1 activity in both APC and T cells contributes to T cell functional polarization during antigen presentation.
Metabolic changes in cells that participate in inflammation, such as activated macrophages and T-helper 17 cells, include a shift towards enhanced glucose uptake, glycolysis and increased activity of the pentose phosphate pathway. Opposing roles in these changes for hypoxia-inducible factor 1α and AMP-activated protein kinase have been proposed. By contrast, anti-inflammatory cells, such as M2 macrophages, regulatory T cells and quiescent memory T cells, have lower glycolytic rates and higher levels of oxidative metabolism. Some anti-inflammatory agents might act by inducing, through activation of AMP-activated protein kinase, a state akin to pseudo-starvation. Altered metabolism may thus participate in the signal-directed programs that promote or inhibit inflammation.
—Recent clinical and morphological evidence established that adrenoleukodystrophy is a distinct X-linked genetic disorder. Fatty acid compositions of lipids in the brain, adrenal and serum from seven patients were examined. Cholesterol esters of both brain and adrenal contained substantial proportions of fatty acids longer than C22 (11.8–41.9% of total in the brain and 13.4-34.8% of total in the adrenal), while cholesterol esters from normal and pathological control specimens contained very little. These very long chain fatty acids were generally saturated in brain cholesterol esters but significant amounts of unsaturated long chain fatty acids were also present in adrenal cholesterol esters. The long chain fatty acids showed bell-shaped distribution with C25 or C26 at the peak. Ganglio-sides from patients’white matter also showed increased proportions of very long-chain fatty acids, up to 50% of the total. Qualitatively similar but much milder fatty acid abnormalities were also found in galactosylceramide of the brain. On the other hand, fatty acids and fatty aldehydes of brain glycerophospholipids, adrenal free fatty acids, triglycerides and glycerophospholipids were not abnormal. Furthermore, serum cholesterol esters from two patients did not show the long-chain fatty acid abnormality found in brain and adrenal cholesterol esters. Sequential extractions with acetone and hexane established that the characteristic birefringent material in the brain and adrenal is indeed cholesterol esters with very long chain fatty acids. This type of fatty acid abnormality has not been described in other pathological conditions and may well represent the unique biochemical abnormality that is directly related to the fundamental genetic defect underlying adrenoleukodystrophy.
Changes in fatty acid composition of complex lipids were analyzed in postmortem white matter from a patient with late onset adrenoleukodystrophy (ALD). The specimen showed three regions with progressive myelin breakdown: morphologically normal white matter; areas with active demyelination and perivascular lymphocyte and macrophage infiltration; and areas with marked gliosis. In the morphologically intact region, cholesterol esters were similar in amount and fatty acid composition to those in control tissue, although marked changes were observed in the actively demyelinating area. Galactolipids in these areas were also similar to those in controls. In contrast, glycerophospholipids were increased in amount and in very long chain fatty acids (VLCFA), which are the hallmark of ALD, at the active edge of the demyelinative lesion and even in the apparently intact sample. Further fractionation of the glycerophospolipids by high performance liquid chromatography showed a significant (up to 39-fold) accumulation of hexacosanoic acid (C26:0) in phosphatidylcholine, but not in other phosphatidyl derivatives. The consistent increases in phosphatidylcholine VLCFA in all samples from the ALD brain, which are postulated to represent progressive stages in the development of the disorder, suggest that phosphatidylcholine may be involved in antigen formation and may underlie an immunological basis for the pathogenesis of ALD.
Anti-myelin immunity is commonly thought to drive multiple sclerosis, yet the initial trigger of this autoreactivity remains elusive. One of the proposed factors for initiating this disease is the primary death of oligodendrocytes. To specifically test such oligodendrocyte death as a trigger for anti-CNS immunity, we inducibly killed oligodendrocytes in an in vivo mouse model. Strong microglia-macrophage activation followed oligodendrocyte death, and myelin components in draining lymph nodes made CNS antigens available to lymphocytes. However, even conditions favoring autoimmunity-bystander activation, removal of regulatory T cells, presence of myelin-reactive T cells and application of demyelinating antibodies-did not result in the development of CNS inflammation after oligodendrocyte death. In addition, this lack of reactivity was not mediated by enhanced myelin-specific tolerance. Thus, in contrast with previously reported impairments of oligodendrocyte physiology, diffuse oligodendrocyte death alone or in conjunction with immune activation does not trigger anti-CNS immunity.
X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disease resulting from mutations in the gene ABCD1 and alterations in peroxisomal beta-oxidation of long chain fatty acids. As it has been frequently discussed, it manifests a wide range of phenotypes in male, with progressive myelopathy being the most common. Even though the gene is localized to the X-chromosome and a region subject to X-inactivation, female carriers still are affected significantly by this condition. It has been stated that between 20 and 50% of women who are carriers may manifest some symptoms and recent evidence has suggested the differences in disease manifestations and relative rates of progression between men and women. However there have been only limited studies specifically addressing this and to date there has been no comprehensive review discussing the different phenotypes in female carriers, as well as the differences in disease onset, progression, disability, nervous system pathology and neuroimaging patterns compared to affected males. This is of key importance as similarities and differences between genders will assist in determining how best to target therapies in all affected individuals as opportunities for treatment present themselves. As will be further addressed in this review, we need to improve our understanding of the associations of emergent neuroimaging techniques to physical disability in this population. We reviewed the clinical presentations in the carrier population, the distinct disability profile and neuroimaging findings in order to put together pieces of this neglected segment in X-ALD and give direction to further studies.
Because of a lack of an appropriate animal model system and the inaccessibility of human oligodendrocytes in vivo, X-linked adrenoleukodystrophy (X-ALD)-induced pluripotent stem cells (iPSCs) would provide a unique cellular model for studying etiopathophysiology and development of therapeutics for X-ALD.
We generated and characterized iPSCs of the 2 major types of X-ALD, childhood cerebral ALD (CCALD) and adrenomyeloneuropathy (AMN), and differentiated them into oligodendrocytes and neurons. We evaluated disease-relevant phenotypes by pharmacological and genetic approaches.
We established iPSCs from the patients with CCALD and AMN. Both CCALD and AMN iPSCs normally differentiated into oligodendrocytes, the cell type primarily affected in the X-ALD brain, indicating no developmental defect due to the ABCD1 mutations. Although low in X-ALD iPSCs, very long chain fatty acid (VLCFA) level was significantly increased after oligodendrocyte differentiation. VLCFA accumulation was much higher in CCALD oligodendrocytes than AMN oligodendrocytes but was not significantly different between CCALD and AMN neurons, indicating that the severe clinical manifestations in CCALD might be associated with abnormal VLCFA accumulation in oligodendrocytes. Furthermore, the abnormal accumulation of VLCFA in the X-ALD oligodendrocytes can be reduced by the upregulated ABCD2 gene expression after treatment with lovastatin or 4-phenylbutyrate.
X-ALD iPSC model recapitulates the key events of disease development (ie, VLCFA accumulation in oligodendrocytes), provides new clues for better understanding of the disease, and allows for early and accurate diagnosis of the disease subtypes. X-ALD oligodendrocytes can be a useful cell model system to develop new therapeutics for treating X-ALD.
Allogeneic hematopoietic stem cell transplantation (HSCT) is the only therapeutic approach that can arrest cerebral demyelination of X-linked adrenoleukodystrophy (ALD) in boys and results in long-term in a good quality of life, provided the procedure is performed at an early stage of disease. Similar benefits of allogeneic HSCT have been demonstrated in adults with cerebral ALD. However, it is not yet known whether allogeneic HSCT can prevent or rescue adrenomyeloneuropathy. Allogeneic HSCT remains associated with significant morbidity and mortality risks, particularly in adults, and not all ALD patients have donors despite the availability of cord blood. The absence of biological markers that can predict the evolutivity of cerebral disease is a major limitation to propose in due time allogeneic HSCT to ALD patients. Recently, HSC gene therapy using lentiviral vector was shown to have comparable efficacy than allogeneic HSCT in two boys with cerebral ALD who had no Human-leukocyte-antigen (HLA)-matched donor. If these results are confirmed in an extended series of patients, HSC gene therapy may become the first therapeutic option for all ALD male patients who develop cerebral demyelination.
X-adrenoleukodystrophy (X-ALD) is a complex disease where inactivation of ABCD1 gene results in clinically diverse phenotypes, the fatal disorder of cerebral ALD (cALD) or a milder disorder of adrenomyeloneuropathy (AMN). Loss of ABCD1 function results in defective beta oxidation of very long chain fatty acids (VLCFA) resulting in excessive accumulation of VLCFA, the biochemical "hall mark" of X-ALD. At present, the ABCD1-mediated mechanisms that determine the different phenotype of X-ALD are not well understood. The studies reviewed here suggest for a "three-hit hypothesis" for neuropathology of cALD. An improved understanding of the molecular mechanisms associated with these three phases of cALD disease should facilitate the development of effective pharmacological therapeutics for X-ALD.
Ablation of functional peroxisomes from all neural cells in Nestin-Pex5 knockout mice caused remarkable neurological abnormalities including motoric and cognitive malfunctioning accompanied by demyelination, axonal degeneration, and gliosis. An oligodendrocyte selective Cnp-Pex5 knockout mouse model shows a similar pathology, but with later onset and slower progression. Until now, the link between these neurological anomalies and the known metabolic alterations, namely the accumulation of very long-chain fatty acids (VLCFA) and reduction of plasmalogens, has not been established. We now focused on the role of peroxisomes in neurons and astrocytes. A neuron-specific peroxisome knockout model, NEX-Pex5, showed neither microscopic nor metabolic abnormalities indicating that the lack of functional peroxisomes within neurons does not cause axonal damage. Axonal integrity and normal behavior was also preserved when peroxisomes were deleted from astrocytes in GFAP-Pex5(-/-) mice. Nevertheless, peroxisomal metabolites were dysregulated in brain including a marked accumulation of VLCFA and a slight reduction in plasmalogens. Interestingly, despite minor targeting of oligodendrocytes in GFAP-Pex5(-/-) mice, these metabolic perturbations were also present in isolated myelin indicating that peroxisomal metabolites are shuttled between different brain cell types. We conclude that absence of peroxisomal metabolism in neurons and astrocytes does not provoke the neurodegenerative phenotype observed after deleting peroxisomes from oligodendrocytes. Lack of peroxisomal metabolism in astrocytes causes increased VLCFA levels in myelin, but this has no major impact on neurological functioning.