Role of Basal Forebrain Neurons in Adrenomyeloneuropathy in Mice and Humans.

OBJECTIVE: X-linked adrenoleukodystrophy is caused by mutations in the peroxisomal half-transporter ABCD1. The most common manifestation is adrenomyeloneuropathy, a hereditary spastic paraplegia of adulthood. The present study set out to understand the role of neuronal ABCD1 in mice and humans with adrenomyeloneuropathy. METHODS: Neuronal expression of ABCD1 during development was assessed in mice and humans. ABCD1-deficient mice and human brain tissues were examined for corresponding pathology. Next, we silenced ABCD1 in cholinergic Sh-sy5y neurons to investigate its impact on neuronal function. Finally, we tested adeno-associated virus vector-mediated ABCD1 delivery to the brain in mice with adrenomyeloneuropathy. RESULTS: ABCD1 is highly expressed in neurons located in the periaqueductal gray matter, basal forebrain and hypothalamus. In ABCD1-deficient mice (Abcd1-/y), these structures showed mild accumulations of α-synuclein. Similarly, healthy human controls had high expression of ABCD1 in deep gray nuclei, whereas X-ALD patients showed increased levels of phosphorylated tau, gliosis, and complement activation in those same regions, albeit not to the degree seen in neurodegenerative tauopathies. Silencing ABCD1 in Sh-sy5y neurons impaired expression of functional proteins and decreased acetylcholine levels, similar to observations in plasma of Abcd1-/y mice. Notably, hind limb clasping in Abcd1-/y mice was corrected through transduction of ABCD1 in basal forebrain neurons following intracerebroventricular gene delivery. INTERPRETATION: Our study suggests that the basal forebrain-cortical cholinergic pathway may contribute to dysfunction in adrenomyeloneuropathy. Rescuing peroxisomal transport activity in basal forebrain neurons and supporting glial cells might represent a viable therapeutic strategy. ANN NEUROL 2024;95:442-458.

Evaluating the strength of evidence for genes implicated in peroxisomal disorders using the ClinGen clinical validity framework and providing updates to the peroxisomal disease nomenclature.

Peroxisomal disorders are heterogeneous in nature, with phenotypic overlap that is indistinguishable without molecular testing. Newborn screening and gene sequencing for a panel of genes implicated in peroxisomal diseases are critical tools for the early and accurate detection of these disorders. It is therefore essential to evaluate the clinical validity of the genes included in sequencing panels for peroxisomal disorders. The Peroxisomal Gene Curation Expert Panel (GCEP) assessed genes frequently included on clinical peroxisomal testing panels using the Clinical Genome Resource (ClinGen) gene-disease validity curation framework and classified gene-disease relationships as Definitive, Strong, Moderate, Limited, Disputed, Refuted, or No Known Disease Relationship. Subsequent to gene curation, the GCEP made recommendations to update the disease nomenclature and ontology in the Monarch Disease Ontology (Mondo) database. Thirty-six genes were assessed for the strength of evidence supporting their role in peroxisomal disease, leading to 36 gene-disease relationships, after two genes were removed for their lack of a role in peroxisomal disease and two genes were curated for two different disease entities each. Of these, 23 were classified as Definitive (64%), one as Strong (3%), eight as Moderate (23%), two as Limited (5%), and two as No known disease relationship (5%). No contradictory evidence was found to classify any relationships as Disputed or Refuted. The gene-disease relationship curations are publicly available on the ClinGen website (https://clinicalgenome.org/affiliation/40049/). The changes to peroxisomal disease nomenclature are displayed on the Mondo website (http://purl.obolibrary.org/obo/MONDO_0019053). The Peroxisomal GCEP-curated gene-disease relationships will inform clinical and laboratory diagnostics and enhance molecular testing and reporting. As new data will emerge, the gene-disease classifications asserted by the Peroxisomal GCEP will be re-evaluated periodically.

Structural Characterization and Quantitation of Ether-Linked Glycerophospholipids in Peroxisome Biogenesis Disorder Tissue by Ultraviolet Photodissociation Mass Spectrometry.

The biological impact of ether glycerophospholipids (GP) in peroxisomal disorders and other diseases makes them significant targets as biomarkers for diagnostic assays or deciphering pathology of the disorders. Ether lipids include both plasmanyl and plasmenyl lipids, which each contain an ether or a vinyl ether bond at the sn-1 linkage position, respectively. This linkage, in contrast to traditional diacyl GPs, precludes their detailed characterization by mass spectrometry via traditional collisional-based MS/MS techniques. Additionally, the isomeric nature of plasmanyl and plasmenyl pairs of ether lipids introduces a further level of complexity that impedes analysis of these species. Here, we utilize 213 nm ultraviolet photodissociation mass spectrometry (UVPD-MS) for detailed characterization of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) plasmenyl and plasmanyl lipids in mouse brain tissue. 213 nm UVPD-MS enables the successful differentiation of these four ether lipid subtypes for the first time. We couple this UVPD-MS methodology to reversed-phase liquid chromatography (RPLC) for characterization and relative quantitation of ether lipids from normal and diseased (Pex7 deficiency modeling the peroxisome biogenesis disorder, RCDP) mouse brain tissue, highlighting the ability to pinpoint specific structural features of ether lipids that are important for monitoring aberrant lipid metabolism in peroxisomal disorders.

Drug discovery for X-linked adrenoleukodystrophy: An unbiased screen for compounds that lower very long-chain fatty acids.

X-linked adrenoleukodystrophy (XALD) is a genetic neurologic disorder with multiple phenotypic presentations and limited therapeutic options. The childhood cerebral phenotype (CCALD), a fatal demyelinating disorder affecting about 35% of patients, and the adult-onset adrenomyeloneuropathy (AMN), a peripheral neuropathy affecting 40%-45% of patients, are both caused by mutations in the ABCD1 gene. Both phenotypes are characterized biochemically by elevated tissue and plasma levels of saturated very long-chain fatty acids (VLCFA), and an increase in plasma cerotic acid (C26:0), along with the clinical presentation, is diagnostic. Administration of oils containing monounsaturated fatty acids, for example, Lorenzo's oil, lowers patient VLCFA levels and reduced the frequency of development of CCALD in presymptomatic boys. However, this therapy is not currently available. Hematopoietic stem cell transplant and gene therapy remain viable therapies for boys with early progressive cerebral disease. We asked whether any existing approved drugs can lower VLCFA and thus open new therapeutic possibilities for XALD. Using SV40-transformed and telomerase-immortalized skin fibroblasts from an XALD patient, we conducted an unbiased screen of a library of approved drugs and natural products for their ability to decrease VLCFA, using measurement of C26:0 in lysophosphatidyl choline (C26-LPC) by tandem mass spectrometry as the readout. While several candidate drugs were initially identified, further testing in primary fibroblast cell lines from multiple CCALD and AMN patients narrowed the list to one drug, the anti-hypertensive drug irbesartan. In addition to lowering C26-LPC, levels of C26:0 and C28:0 in total fibroblast lipids were reduced. The effect of irbesartan was dose dependent between 2 and 10 µM. When male XALD mice received orally administered irbesartan at a dose of 10 mg/kg/day, there was no reduction in plasma C26-LPC. However, irbesartan failed to lower mouse fibroblast C26-LPC consistently. The results of these studies indicate a potential therapeutic benefit of irbesartan in XALD that should be validated by further study.

MRI surveillance of boys with X-linked adrenoleukodystrophy identified by newborn screening: Meta-analysis and consensus guidelines.

BACKGROUND: Among boys with X-Linked adrenoleukodystrophy, a subset will develop childhood cerebral adrenoleukodystrophy (CCALD). CCALD is typically lethal without hematopoietic stem cell transplant before or soon after symptom onset. We sought to establish evidence-based guidelines detailing the neuroimaging surveillance of boys with neurologically asymptomatic adrenoleukodystrophy. METHODS: To establish the most frequent age and diagnostic neuroimaging modality for CCALD, we completed a meta-analysis of relevant studies published between January 1, 1970 and September 10, 2019. We used the consensus development conference method to incorporate the resulting data into guidelines to inform the timing and techniques for neuroimaging surveillance. Final guideline agreement was defined as >80% consensus. RESULTS: One hundred twenty-three studies met inclusion criteria yielding 1285 patients. The overall mean age of CCALD diagnosis is 7.91 years old. The median age of CCALD diagnosis calculated from individual patient data is 7.0 years old (IQR: 6.0-9.5, n = 349). Ninety percent of patients were diagnosed between 3 and 12. Conventional MRI was most frequently reported, comprised most often of T2-weighted and contrast-enhanced T1-weighted MRI. The expert panel achieved 95.7% consensus on the following surveillance parameters: (a) Obtain an MRI between 12 and 18 months old. (b) Obtain a second MRI 1 year after baseline. (c) Between 3 and 12 years old, obtain a contrast-enhanced MRI every 6 months. (d) After 12 years, obtain an annual MRI. CONCLUSION: Boys with adrenoleukodystrophy identified early in life should be monitored with serial brain MRIs during the period of highest risk for conversion to CCALD.

Associations Between Systemic Omega-3 Fatty Acid Levels With Moderate-to-Severe Dry Eye Disease Signs and Symptoms at Baseline in the Dry Eye Assessment and Management Study.

PURPOSE: Omega-3 (n-3) fatty acid supplementation is used to treat systemic inflammatory diseases, but the role of n-3 in the pathophysiology and therapy of dry eye disease (DED) is not definitive. We evaluated the relationship of systemic n-3 levels with signs and symptoms at baseline in the Dry Eye Assessment and Management (DREAM) Study. METHODS: Blood samples from participants at baseline were analyzed for n-3 and n-6, measured as relative percentage by weight among all fatty acids in erythrocytes. Symptoms were evaluated using the Ocular Surface Disease Index. Signs including conjunctival staining, corneal staining, tear breakup time (TBUT), and Schirmer's test with anesthesia were also evaluated. RESULTS: There was no correlation between the systemic n-3 levels and DED symptoms. When the associations with signs of DED were assessed, lower DHA levels were associated with higher conjunctival staining, with mean scores of 3.31, 2.96, and 2.82 for low, medium, and high levels of DHA, respectively (linear trend P=0.007). None of the other signs were associated with DHA or the other measures of n-3. CONCLUSION: Previous studies have found varying results on the role of n-3 supplementation with the signs and symptoms of DED. Among patients with DED enrolled in the DREAM Study, lower systemic n-3 levels were not associated with worse symptoms and most signs of DED.

Allelic Expression Imbalance Promoting a Mutant PEX6 Allele Causes Zellweger Spectrum Disorder.

Zellweger spectrum disorders (ZSDs) are autosomal-recessive disorders that are caused by defects in peroxisome biogenesis due to bi-allelic mutations in any of 13 different PEX genes. Here, we identified seven unrelated individuals affected with an apparent dominant ZSD in whom a heterozygous mutant PEX6 allele (c.2578C>T [p.Arg860Trp]) was overrepresented due to allelic expression imbalance (AEI). We demonstrated that AEI of PEX6 is a common phenomenon and is correlated with heterozygosity for a frequent variant in the 3' untranslated region (UTR) of the mutant allele, which disrupts the most distal of two polyadenylation sites. Asymptomatic parents, who were heterozygous for PEX c.2578C>T, did not show AEI and were homozygous for the 3' UTR variant. Overexpression models confirmed that the overrepresentation of the pathogenic PEX6 c.2578T variant compared to wild-type PEX6 c.2578C results in a peroxisome biogenesis defect and thus constitutes the cause of disease in the affected individuals. AEI promoting the overrepresentation of a mutant allele might also play a role in other autosomal-recessive disorders, in which only one heterozygous pathogenic variant is identified.

Peroxisomal biogenesis is genetically and biochemically linked to carbohydrate metabolism in Drosophila and mouse.

Peroxisome biogenesis disorders (PBD) are a group of multi-system human diseases due to mutations in the PEX genes that are responsible for peroxisome assembly and function. These disorders lead to global defects in peroxisomal function and result in severe brain, liver, bone and kidney disease. In order to study their pathogenesis we undertook a systematic genetic and biochemical study of Drosophila pex16 and pex2 mutants. These mutants are short-lived with defects in locomotion and activity. Moreover these mutants exhibit severe morphologic and functional peroxisomal defects. Using metabolomics we uncovered defects in multiple biochemical pathways including defects outside the canonical specialized lipid pathways performed by peroxisomal enzymes. These included unanticipated changes in metabolites in glycolysis, glycogen metabolism, and the pentose phosphate pathway, carbohydrate metabolic pathways that do not utilize known peroxisomal enzymes. In addition, mutant flies are starvation sensitive and are very sensitive to glucose deprivation exhibiting dramatic shortening of lifespan and hyperactivity on low-sugar food. We use bioinformatic transcriptional profiling to examine gene co-regulation between peroxisomal genes and other metabolic pathways and we observe that the expression of peroxisomal and carbohydrate pathway genes in flies and mouse are tightly correlated. Indeed key steps in carbohydrate metabolism were found to be strongly co-regulated with peroxisomal genes in flies and mice. Moreover mice lacking peroxisomes exhibit defective carbohydrate metabolism at the same key steps in carbohydrate breakdown. Our data indicate an unexpected link between these two metabolic processes and suggest metabolism of carbohydrates could be a new therapeutic target for patients with PBD.

Adaptive variation, including local adaptation, requires decades to become evident in common gardens.

Population-level adaptation to spatial variation in factors such as climate and soils is critical for climate-vulnerability assessments, restoration seeding, and other ecological applications in species management, and the underlying information is typically based on common-garden studies that are short duration. Here, we show >20 yr were required for adaptive differences to emerge among 13 populations of a widespread shrub (sagebrush, Artemisia tridentata ssp wyomingensis) collected from around the western United States and planted into common gardens. Additionally, >10 yr were required for greater survival of local populations, that is, local adaptation, to become evident. Variation in survival was best explained by the combination of populations' home ecoregion combined with grouping of minimum temperature and aridity. Additional reductions in survival were explained by ungrouped (i.e., continuous) measures of garden-to-population-origin separation in geographic distance (5% decrease in survival per 100 km increase in separation; R2  = 0.22) and especially in minimum temperature in younger plants (-4% per + °C difference, R2  = 0.56 vs. 0.29 in the 14th vs. 27th post-planting years, respectively). Longer-term common garden studies are needed. While we await them, uncertainty in adaptive variation resulting from short-term observations could be quantitatively estimated and reported with seed-transfer guidelines to reduce risks of introducing maladapted provenances in restoration.

A metabolomic map of Zellweger spectrum disorders reveals novel disease biomarkers.

PURPOSE: Peroxisome biogenesis disorders-Zellweger spectrum disorders (PBD-ZSD) are metabolic diseases with multisystem manifestations. Individuals with PBD-ZSD exhibit impaired peroxisomal biochemical functions and have abnormal levels of peroxisomal metabolites, but the broader metabolic impact of peroxisomal dysfunction and the utility of metabolomic methods is unknown. METHODS: We studied 19 individuals with clinically and molecularly characterized PBD-ZSD. We performed both quantitative peroxisomal biochemical diagnostic studies in parallel with untargeted small molecule metabolomic profiling in plasma samples with detection of >650 named compounds. RESULTS: The cohort represented intermediate to mild PBD-ZSD subjects with peroxisomal biochemical alterations on targeted analysis. Untargeted metabolomic profiling of these samples revealed elevations in pipecolic acid and long-chain lysophosphatidylcholines, as well as an unanticipated reduction in multiple sphingomyelin species. These sphingomyelin reductions observed were consistent across the PBD-ZSD samples and were rare in a population of >1,000 clinical samples. Interestingly, the pattern or "PBD-ZSD metabolome" was more pronounced in younger subjects suggesting studies earlier in life reveal larger biochemical changes. CONCLUSION: Untargeted metabolomics is effective in detecting mild to intermediate cases of PBD-ZSD. Surprisingly, dramatic reductions in plasma sphingomyelin are a consistent feature of the PBD-ZSD metabolome. The use of metabolomics in PBD-ZSD can provide insight into novel biomarkers of disease.

A novel bile acid biosynthesis defect due to a deficiency of peroxisomal ABCD3.

ABCD3 is one of three ATP-binding cassette (ABC) transporters present in the peroxisomal membrane catalyzing ATP-dependent transport of substrates for metabolic pathways localized in peroxisomes. So far, the precise function of ABCD3 is not known. Here, we report the identification of the first patient with a defect of ABCD3. The patient presented with hepatosplenomegaly and severe liver disease and showed a striking accumulation of peroxisomal C27-bile acid intermediates in plasma. Investigation of peroxisomal parameters in skin fibroblasts revealed a reduced number of enlarged import-competent peroxisomes. Peroxisomal beta-oxidation of C26:0 was normal, but beta-oxidation of pristanic acid was reduced. Genetic analysis revealed a homozygous deletion at the DNA level of 1758bp, predicted to result in a truncated ABCD3 protein lacking the C-terminal 24 amino acids (p.Y635NfsX1). Liver disease progressed and the patient required liver transplantation at 4 years of age but expired shortly after transplantation. To corroborate our findings in the patient, we studied a previously generated Abcd3 knockout mouse model. Abcd3-/- mice accumulated the branched chain fatty acid phytanic acid after phytol loading. In addition, analysis of bile acids revealed a reduction of C24 bile acids, whereas C27-bile acid intermediates were significantly increased in liver, bile and intestine of Abcd3-/- mice. Thus, both in the patient and in Abcd3-/- mice, there was evidence of a bile acid biosynthesis defect. In conclusion, our studies show that ABCD3 is involved in transport of branched-chain fatty acids and C27 bile acids into the peroxisome and that this is a crucial step in bile acid biosynthesis.

X-linked adrenoleukodystrophy in a chimpanzee due to an ABCD1 mutation reported in multiple unrelated humans.

BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder leading to the accumulation of very long chain fatty acids (VLCFA) due to a mutation in the ABCD1 gene. ABCD1 mutations lead to a variety of phenotypes, including cerebral X-ALD and adrenomyeloneuropathy (AMN) in affected males and 80% of carrier females. There is no definite genotype-phenotype correlation with intrafamilial variability. Cerebral X-ALD typically presents in childhood, but can also present in juveniles and adults. The most affected tissues are the white matter of the brain and adrenal cortex. MRI demonstrates a characteristic imaging appearance in cerebral X-ALD that is used as a diagnostic tool. OBJECTIVES: We aim to correlate a mutation in the ABCD1 gene in a chimpanzee to the human disease X-ALD based on MRI features, neurologic symptoms, and plasma levels of VLCFA. METHODS: Diagnosis of X-ALD made using MRI, blood lipid profiling, and DNA sequencing. RESULTS: An 11-year-old chimpanzee showed remarkably similar features to juvenile onset cerebral X-ALD in humans including demyelination of frontal lobes and corpus callosum on MRI, elevated plasma levels of C24:0 and C26:0, and identification of the c.1661G>A ABCD1 variant. CONCLUSIONS: This case study presents the first reported case of a leukodystrophy in a great ape, and underscores the fidelity of MRI pattern recognition in this disorder across species.

Therapeutic strategies in adrenoleukodystrophy.

Adrenoleukodystrophy (ALD) is an X­linked hereditary disorder due to mutations of the ABCD1 gene, which encodes a peroxisomal transport protein necessary for very long-chain fatty acid degradation (VLCFA). Toxic accumulation thereof is associated with a proinflammatory state and eventual cell death in multiple tissues. ALD may manifest either as a fatal, rapidly progressive demyelinating disease in boys and adult men, or as a slowly progressive adult-onset long-tract myelopathy along with peripheral neuropathy. Our understanding of manifold mechanisms implicated in the disease pathology is currently incomplete, as neither genotype-phenotype correlation nor the trigger for cerebral disease has been described. Therapy objectives are therefore broadly aimed at correcting either the gene mutation or downstream molecular effects, such as oxidative stress. Advancements in disease detection, including the newly implemented newborn screening in the US and imaging modalities, allow for more timely intervention in the form of hematopoietic stem cell transplantation (HSCT), which may only be performed in early cerebral disease states.

Homeostasis of phospholipids - The level of phosphatidylethanolamine tightly adapts to changes in ethanolamine plasmalogens.

Ethanolamine plasmalogens constitute a group of ether glycerophospholipids that, due to their unique biophysical and biochemical properties, are essential components of mammalian cellular membranes. Their importance is emphasized by the consequences of defects in plasmalogen biosynthesis, which in humans cause the fatal disease rhizomelic chondrodysplasia punctata (RCDP). In the present lipidomic study, we used fibroblasts derived from RCDP patients, as well as brain tissue from plasmalogen-deficient mice, to examine the compensatory mechanisms of lipid homeostasis in response to plasmalogen deficiency. Our results show that phosphatidylethanolamine (PE), a diacyl glycerophospholipid, which like ethanolamine plasmalogens carries the head group ethanolamine, is the main player in the adaptation to plasmalogen insufficiency. PE levels were tightly adjusted to the amount of ethanolamine plasmalogens so that their combined levels were kept constant. Similarly, the total amount of polyunsaturated fatty acids (PUFAs) in ethanolamine phospholipids was maintained upon plasmalogen deficiency. However, we found an increased incorporation of arachidonic acid at the expense of docosahexaenoic acid in the PE fraction of plasmalogen-deficient tissues. These data show that under conditions of reduced plasmalogen levels, the amount of total ethanolamine phospholipids is precisely maintained by a rise in PE. At the same time, a shift in the ratio between ω-6 and ω-3 PUFAs occurs, which might have unfavorable, long-term biological consequences. Therefore, our findings are not only of interest for RCDP but may have more widespread implications also for other disease conditions, as for example Alzheimer's disease, that have been associated with a decline in plasmalogens.

Peroxisome biogenesis disorders in the Zellweger spectrum: An overview of current diagnosis, clinical manifestations, and treatment guidelines.

Peroxisome biogenesis disorders in the Zellweger spectrum (PBD-ZSD) are a heterogeneous group of genetic disorders caused by mutations in PEX genes responsible for normal peroxisome assembly and functions. As a result of impaired peroxisomal activities, individuals with PBD-ZSD can manifest a complex spectrum of clinical phenotypes that typically result in shortened life spans. The extreme variability in disease manifestation ranging from onset of profound neurologic symptoms in newborns to progressive degenerative disease in adults presents practical challenges in disease diagnosis and medical management. Recent advances in biochemical methods for newborn screening and genetic testing have provided unprecedented opportunities for identifying patients at the earliest possible time and defining the molecular bases for their diseases. Here, we provide an overview of current clinical approaches for the diagnosis of PBD-ZSD and provide broad guidelines for the treatment of disease in its wide variety of forms. Although we anticipate future progress in the development of more effective targeted interventions, the current guidelines are meant to provide a starting point for the management of these complex conditions in the context of personalized health care.

A model-based approach to assess the exposure-response relationship of Lorenzo's oil in adrenoleukodystrophy.

AIMS: X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder, most commonly affecting boys, associated with increased very long chain fatty acids (C26:0) in all tissues, causing cerebral demyelination and adrenocortical insufficiency. Certain monounsaturated long chain fatty acids including oleic and erucic acids, known as Lorenzo's oil (LO), lower plasma C26:0 levels. The aims of this study were to characterize the effect of LO administration on plasma C26:0 concentrations and to determine whether there is an association between plasma concentrations of erucic acid or C26:0 and the likelihood of developing brain MRI abnormalities in asymptomatic boys. METHODS: Non-linear mixed effects modelling was performed on 2384 samples collected during an open label single arm trial. The subjects (n = 104) were administered LO daily at ~2-3 mg kg(-1) with a mean follow-up of 4.88 ± 2.76 years. The effect of erucic acid exposure on plasma C26:0 concentrations was characterized by an inhibitory fractional Emax model. A Weibull model was used to characterize the time-to-developing MRI abnormality. RESULTS: The population estimate for the fractional maximum reduction of C26:0 plasma concentrations was 0.76 (bootstrap 95% CI 0.73, 0.793). Our time-to-event analyses showed that every mg l(-1) increase in time-weighted average of erucic acid and C26:0 plasma concentrations was, respectively, associated with a 3.7% reduction and a 753% increase in the hazard of developing MRI abnormality. However, the results were not significant (P = 0.5344, 0.1509, respectively). CONCLUSIONS: LO administration significantly reduces the abnormally high plasma C26:0 concentrations in X-ALD patients. Further studies to evaluate the effect of LO on the likelihood of developing brain MRI abnormality are warranted.

Brain endothelial dysfunction in cerebral adrenoleukodystrophy.

See Aubourg (doi:10.1093/awv271) for a scientific commentary on this article.X-linked adrenoleukodystrophy is caused by mutations in the ABCD1 gene leading to accumulation of very long chain fatty acids. Its most severe neurological manifestation is cerebral adrenoleukodystrophy. Here we demonstrate that progressive inflammatory demyelination in cerebral adrenoleukodystrophy coincides with blood-brain barrier dysfunction, increased MMP9 expression, and changes in endothelial tight junction proteins as well as adhesion molecules. ABCD1, but not its closest homologue ABCD2, is highly expressed in human brain microvascular endothelial cells, far exceeding its expression in the systemic vasculature. Silencing of ABCD1 in human brain microvascular endothelial cells causes accumulation of very long chain fatty acids, but much later than the immediate upregulation of adhesion molecules and decrease in tight junction proteins. This results in greater adhesion and transmigration of monocytes across the endothelium. PCR-array screening of human brain microvascular endothelial cells after ABCD1 silencing revealed downregulation of both mRNA and protein levels of the transcription factor c-MYC (encoded by MYC). Interestingly, MYC silencing mimicked the effects of ABCD1 silencing on CLDN5 and ICAM1 without decreasing the levels of ABCD1 protein itself. Together, these data demonstrate that ABCD1 deficiency induces significant alterations in brain endothelium via c-MYC and may thereby contribute to the increased trafficking of leucocytes across the blood-brain barrier as seen in cerebral adrenouleukodystrophy.

Adenoassociated virus serotype 9-mediated gene therapy for x-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is a devastating neurological disorder caused by mutations in the ABCD1 gene that encodes a peroxisomal ATP-binding cassette transporter (ABCD1) responsible for transport of CoA-activated very long-chain fatty acids (VLCFA) into the peroxisome for degradation. We used recombinant adenoassociated virus serotype 9 (rAAV9) vector for delivery of the human ABCD1 gene (ABCD1) to mouse central nervous system (CNS). In vitro, efficient delivery of ABCD1 gene was achieved in primary mixed brain glial cells from Abcd1-/- mice as well as X-ALD patient fibroblasts. Importantly, human ABCD1 localized to the peroxisome, and AAV-ABCD1 transduction showed a dose-dependent effect in reducing VLCFA. In vivo, AAV9-ABCD1 was delivered to Abcd1-/- mouse CNS by either stereotactic intracerebroventricular (ICV) or intravenous (IV) injections. Astrocytes, microglia and neurons were the major target cell types following ICV injection, while IV injection also delivered to microvascular endothelial cells and oligodendrocytes. IV injection also yielded high transduction of the adrenal gland. Importantly, IV injection of AAV9-ABCD1 reduced VLCFA in mouse brain and spinal cord. We conclude that AAV9-mediated ABCD1 gene transfer is able to reach target cells in the nervous system and adrenal gland as well as reduce VLCFA in culture and a mouse model of X-ALD.

Dietary influences on tissue concentrations of phytanic acid and AMACR expression in the benign human prostate.

BACKGROUND: Alpha-methylacyl-CoA racemase (AMACR) is an enzyme involved in fatty acid metabolism that is markedly over-expressed in virtually all prostate cancers (PCa), relative to benign tissue. One of AMACR's primary substrates, phytanic acid, is derived predominately from red meat and dairy product consumption. Epidemiological evidence suggests links between dairy/red meat intake, as well as phytanic acid levels, and elevated PCa risk. This study investigates the relationships among dietary intake, serum and tissue concentrations of phytanic acid, and AMACR expression (mRNA and protein) in the histologically benign human prostate. METHODS: Men undergoing radical prostatectomy for the treatment of localized disease provided a food frequency questionnaire (n = 68), fasting blood (n = 35), benign fresh frozen prostate tissue (n = 26), and formalin-fixed paraffin-embedded (FFPE) sections (n = 67). Serum and tissue phytanic acid concentrations were obtained by gas chromatography-mass spectrometry. We extracted RNA from epithelial cells using laser capture microdissection and quantified mRNA expression of AMACR and other genes involved in the peroxisomal phytanic acid metabolism pathway via qRT-PCR. Immunohistochemistry for AMACR was performed on FFPE sections and subsequently quantified via digital image analysis. Associations between diet, serum, and tissue phytanic acid levels, as well as AMACR and other gene expression levels were assessed by partial Spearman correlation coefficients. RESULTS: High-fat dairy intake was the strongest predictor of circulating phytanic acid concentrations (r = 0.35, P = 0.04). Tissue phytanic acid concentrations were not associated with any dietary sources and were only weakly correlated with serum levels (r = 0.29, P = 0.15). AMACR gene expression was not associated with serum phytanic acid (r = 0.13, P = 0.47), prostatic phytanic acid concentrations (r = 0.03, P = 0.88), or AMACR protein expression (r = -0.16, P = 0.20). CONCLUSIONS: Our data underscore the complexity of the relationship between AMACR and its substrates and do not support the unifying hypothesis that excess levels of dietary phytanic acid are responsible for both the overexpression of AMACR in prostate cancer and the potential association between PCa risk and intake of dairy foods and red meat.

Streamlined determination of lysophosphatidylcholines in dried blood spots for newborn screening of X-linked adrenoleukodystrophy.

BACKGROUND: Pre-symptomatic hematopoietic stem cell transplantation is essential to achieve best possible outcomes for patients with the childhood cerebral form of X-linked adrenoleukodystrophy (X-ALD). We describe a high-throughput method for measurement of C20-C26 lysophosphatidylcholines (LPCs) and biochemical diagnosis of X-ALD using the same dried blood spots (DBS) routinely used for newborn screening. METHODS: LPCs are extracted from 3-mm DBS punch with methanol containing an isotopically labeled LPC as internal standard. This extract is transferred to a 96-well plate, evaporated and then reconstituted in mobile phase for flow injection analysis tandem mass spectrometry (FIA-MS/MS) in selected reaction monitoring mode for measurement of four different LPCs (C20, C22, C24, C26) and the internal standard (d4-C26-LPC). Analysis time is 1.5min per sample. RESULTS: The mean CVs from the intra- and inter-assay experiments for LPCs were 6.3-15.1% for C20-LPC, 4.4-18.6% for C22-LPC and 4.5-14.3% for C24-LPC. Limits of detection were determined for C20-LPC (LOD=0.03µg/mL), C22-LPC (0.03µg/mL), C24-LPC (0.03µg/mL) and C26-LPC (0.01µg/mL). Reference ranges were established from DBS of 130 newborns and 20 adults. Samples of patients with X-ALD (n=16), peroxisomal biogenesis disorders (n=8), and X-ALD carriers (n=12) were analyzed blindly and all were correctly identified. CONCLUSION: Analysis of LPC species by FIA-MS/MS is a fast, simple and reliable method to screen for X-ALD and other peroxisomal disorders in DBS. To maximize specificity, abnormal results can be verified by a 2nd tier assay using LC-MS/MS.