Peroxisome biogenesis disorders (PBD) affect multiple organ systems. It is characterized by neurological dysfunction, hypotonia, ocular anomalies, craniofacial abnormalities, and absence of peroxisomes in fibroblasts. PBDs are associated with mutations in any of fourteen different PEX genes, which are involved in peroxisome biogenesis. Zellweger spectrum disorder (ZSD) is a severe form of PBD. More than 90% of the ZSD cases have mutations in PEX1, PEX6, PEX10, PEX12, and PEX26. Mutations in the PEX19 gene are rarely associated with PBD/ZSD; however, a large proportion of PEX26 mutations are associated with ZSD.

We recruited two Saudi families with multiple affected individuals with dysmorphic features, including hypertelorism, large open fontanelles, generalized hypotonia, and epicanthal folds with poor reflexes since birth. Whole exome sequencing (WES) and Sanger sequencing was performed to identify the genetic cause. The frequency and pathogenicity of the identified mutations were assessed using various online bioinformatics tools.

WES identified a novel nonsense variant (c.367C > T) in the PEX19 gene in family A patients. This nonsense mutation was predicted to cause premature termination (p.Gln123*). A previously reported synonymous variant (c.228C > T; p.Gly76Gly) in PEX26 was found in a patient from family B. Both variants were segregating in an autosomal recessive manner in the respective families.

The present study has added a novel nonsense mutation to the mutation spectrum of PEX19, which is the second null mutation identified to date. Moreover, in this study, the importance of a synonymous exonic variant of PEX26 close to the splice donor site was explored in relation to pre-mRNA splicing and resulting disease manifestations.

This study aims to evaluate visual function and morphological changes in patients with adrenoleukodystrophy (ALD) through sweep-source optical coherence tomography (SS-OCT) and patterned visual evoked potentials (PVEP), with the goal of providing more accurate and comprehensive data for the diagnosis, monitoring, and assessment of treatment efficacy in patients with ALD.

This study included 46 ALD patients and 44 healthy controls. The thickness of the retinal nerve fiber layer (RNFL) around the optic disc, as well as the macular RNFL and ganglion cell-inner plexiform layer (GCIPL) thickness, was assessed using SS-OCT. The peak time and amplitude of the P100 wave were also measured using PVEP. Factors that were statistically significant in the univariate analysis were subjected to stepwise binary logistic regression analysis for further investigation. Predictive performance was evaluated by constructing receiver operating characteristic (ROC) curves and compared using DeLong's test.

Compared with the control patients, ALD patients presented a significant increase in the peak time of the P100 wave (P < 0.05); however, no notable difference in amplitude was observed (P > 0.05). Additionally, substantial decreases in the RNFL and GCIPL thicknesses were observed within the parafoveal ring, especially in the superior and nasal quadrants (P < 0.05). The area under the curve for the binary logistic stepwise regression model was 0.883, with a sensitivity of 0.95, which surpassed the performance of the individual parameters.

ALD patients present with abnormal retinal structures and a PVEP peak time delay. Combining these two parameters could increase the accuracy of an early ALD diagnosis.

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder caused by mutations in the ABCD1 gene. We reported the clinical features and genetic findings of 17 X-ALD patients. Fifteen variants were identified, including five novel mutations: c.700dupC (p.Arg234Profs*67), c.743G>A (p.Gly248Asp), c.1469_1471delTGG (p.Val490del), c.1577C>A (p.Thr526Lys), and c.1658T>C (p.Leu553Pro), which were reported for the first time in X-ALD patients. Functional analysis confirmed the pathogenicity of novel variants at the protein and subcellular localization level. The p.Arg234Profs*67 mutant protein was undetectable, likely due to NMD-mediated mRNA degradation. This study expands the mutation spectrum and clinical profile of X-ALD, suggesting a potential correlation between the extent of protein dysfunction and disease severity.

Microglia, the immune cells of the central nervous system, activate neuroinflammatory pathways in response to homeostatic disturbances, a process implicated in the pathogenesis of various neurodegenerative diseases. Emerging evidence identifies abnormal microglial activation as a causal factor at the onset of peroxisomal leukodystrophies, including X-linked adrenoleukodystrophy (X-ALD). This study investigates how primary peroxisomal deficiencies influence oxidative properties of microglia and examines the subsequent impact on neurons and oligodendrocytes. Using BV-2 microglial cells lacking ABCD1, ABCD2, or ACOX1, peroxisomal proteins that play key roles in the very-long-chain fatty acid beta-oxidation, we analyzed their response under basal condition and after stimulation by lipopolysaccharide (LPS). Transcriptomic analysis of the mutant microglial cells revealed numerous differentially expressed genes, particularly in redox-related pathways following LPS exposure. These changes are consistent with the increased production of reactive oxygen species (ROS) and nitric oxide (NO). Conditioned media (CM) from the mutant cells were then applied to cultures of neuron and oligodendrocyte cell lines. Exposure to CM from LPS-stimulated mutant microglial cells significantly increased apoptosis in both cell types. Furthermore, treated neurons exhibited a reduction in cell complexity and an increased ability to secrete neuropeptides. These findings demonstrate that peroxisomal impairments in microglia exacerbate inflammatory response and ROS/NO production, affecting the survival of neurons and oligodendrocytes, as well as neuronal morphology and function. This dysfunction might contribute to the early neurodegenerative events in X-ALD by triggering and sustaining neuroinflammatory cascades. Therapeutic strategies that target microglial activation and secretion profiles could hold promise in managing peroxisomal disorders such as X-ALD.

X-linked adrenoleukodystrophy (XALD) can affect the eyes. Existing therapies are hampered by early quantitative examination methods. This study used an optical coherence tomography angiography system (OCTA) to investigate retinal microvascular density and perfusion in XALD patients.

Fifty-two patients and 47 age-matched controls were included in this cross-sectional study. The patients were divided into three groups (symptomatic, less symptomatic, and controls). We compared the foveal avascular zone area, vascular density and perfusion area at the superficial vascular complex (SVC) and deep vascular complex (DVC) of the peripapillary and macular between the groups. We correlated these measurements with scale scores.

Compared with the controls, the symptomatic group had significantly lower vascular density in the superior nasal sector of the peripapillary SVC (MD - 4.940884; 95% CI - 9.655061 to - 0.226707; p = 0.036), lower vascular density (MD - 4.259225; 95% CI - 8.248627  to  - 0.269823; p = 0.032) and lower perfusion area (MD - 0.180304; 95% CI - 0.337135  to  - 0.023472; p = 0.018) in the peripheral ring superior quadrant of the macular SVC. Compared with the less symptomatic group, the symptomatic group exhibited a significantly lower vascular density (MD - 5.635483; 95% CI - 10.450009  to  - 0.820957; p = 0.015) and perfusion area (MD - 0.063351; 95% CI - 0.116611  to  - 0.010091; p = 0.013) in the superior nasal sector of the peripapillary SVC; lower vascular density (MD - 4.817846; 95% CI - 8.924294  to  - 0.711399; p = 0.015) and perfusion area (MD - 0.202707; 95% CI - 0.369499  to  - 0.035915; p = 0.011) in the peripheral ring superior quadrant of the macular SVC; and greater vascular density (MD 7.209401; 95% CI 0.818716-13.600086; p = 0.021) and perfusion area (MD 0.047320; 95% CI 0.001685-0.092956; p = 0.039) in the inferior nasal sector of the peripapillary DVC. Among the 52 patients, the expanded disability status score (EDSS) was moderately negatively correlated with the vascular density (p = 0.001) and perfusion area (p = 0.002) in the peripheral ring superior quadrant of the macular SVC.

Changes in retinal vascular density and perfusion exist in XALD patients and are correlated with disease severity. OCTA has the potential to monitor the progression of XALD.

Many cellular functions are compartmentalized within the optimized environments of organelles. However, processing or storage of metabolites from the same pathway can occur in multiple organelles. Thus, spatially separated organelles need to cooperate functionally. Coordination between organelles in different specialized cells is also needed, with shared metabolites passed via circulation. Peroxisomes are membrane-bounded organelles responsible for cellular redox and lipid metabolism in eukaryotic cells. Peroxisomes coordinate with other organelles including mitochondria, endoplasmic reticulum, lysosomes, and lipid droplets. This functional coordination requires, or is at least enhanced by, direct contact between peroxisomes and other organelles. Peroxisome dysfunction in humans leads to multiorgan effects including neurological, metabolic, developmental, and age-related diseases. Thus, increased understanding of peroxisome coordination with other organelles, especially cells in various organs is essential. Drosophila melanogaster (fruit fly) has emerged recently as an effective animal model for understanding peroxisomes. Here we review current knowledge of pathways regulating coordination between peroxisomes with other organelles in flies, speculating about analogous roles for conserved Drosophila genes encoding proteins with known organelle coordinating roles in other species.

X-linked adrenoleukodystrophy (ALD) is a peroxisomal disorder resulting from pathogenic variants in the ABCD1 gene that primarily affects the nervous system and is characterized by progressive axonal degeneration in the spinal cord and peripheral nerves and leukodystrophy. Dysfunction of peroxisomal very long-chain fatty acid (VLCFA) degradation has been implicated in ALD pathology, but the impact on astrocytes, which critically support neuronal function, remains poorly understood. Fibroblasts from four ALD patients were reprogrammed to generate human-induced pluripotent stem cells (hiPSC). hiPSC-derived astrocytes were generated to study the impact of ALD on astrocytic fatty acid homeostasis. Our study reveals significant changes in the lipidome of ALD hiPSC-derived astrocytes, characterized by an enrichment of VLCFAs across multiple lipid classes, including triacylglycerols, cholesteryl esters, and phosphatidylcholines. Importantly, ALD hiPSC-derived astrocytes not only exhibit intrinsic lipid dysregulation but also affect the dendritic tree complexity of neurons in co-culture systems. These findings highlight the cell-autonomous effects of pathogenic variants in the ABCD1 protein on astrocytes and their microenvironment, shed light on potential mechanisms underlying ALD neuropathology, and underscore the critical role of astrocytes in neuronal health.

Pediatric monogenetic diseases encompass a spectrum of debilitating neurological disorders that affect infants and children, often resulting in profound cognitive and motor impairments. Gene replacement therapy holds immense promise in addressing the underlying genetic defects responsible for these conditions. Adeno-associated virus (AAV) vectors have emerged as a leading platform for delivering therapeutic genes due to their safety profile and ability to transduce various cell types, including neurons. This review highlights recent advancements in AAV-mediated gene replacement therapy for pediatric monogenetic diseases, focusing on key preclinical and clinical studies. We discuss various strategies to enhance transduction efficiency, target specificity, and safety. Furthermore, we explore challenges such as immune responses, along with innovative approaches to overcome these obstacles. Moreover, we examine the clinical outcomes and safety profiles of AAV-based gene therapies in pediatric patients, providing insights into the feasibility and efficacy of these interventions. Finally, we discuss future directions and potential avenues for further research to optimize the therapeutic potential of AAV-delivered gene replacement therapy for pediatric encephalopathies, ultimately aiming to improve the quality of life for affected individuals and their families.

X-linked adrenoleukodystrophy (X-ALD) is a severe genetic disorder caused by ABCD1 mutations, resulting in the buildup of very-long-chain fatty acids, leading to significant neurological decline and adrenal insufficiency. Despite advancements in understanding the mechanisms of X-ALD, its pathophysiology remains incompletely understood, complicating the development of effective treatments. This review provides a comprehensive overview of X-ALD, with a focus on the genetic and biochemical roles of ABCD1 and the impacts of its mutations. Current therapeutic approaches are evaluated, discussing their limitations, and emphasizing the need to fully elucidate the pathogenesis of X-ALD. Additionally, this review highlights the importance of international collaboration to enhance systematic data collection and advance biomarker discovery, ultimately improving patient outcomes with X-ALD.

"Bubblegum" acyl-CoA synthetase (ACSBG1) is a pivotal player in lipid metabolism during mouse brain development, facilitating the activation of long-chain fatty acids (LCFA) and their incorporation into lipid species that are crucial for brain function. ACSBG1 converts LCFA into acyl-CoA derivatives, supporting vital metabolic processes. Fruit fly mutants lacking ACSBG1 exhibited neurodegeneration and had elevated levels of very long-chain fatty acids (VLCFA), characteristics of human X-linked adrenoleukodystrophy (XALD). To explore ACSBG1's function and potential as a therapeutic target in XALD, we created an ACSBG1 knockout (Acsbg1-/-) mouse and examined the effects on brain FA metabolism during development. Phenotypically, Acsbg1-/- mice resembled wild type (w.t.) mice. ACSBG1 expression was found mainly in tissue affected pathologically in XALD, namely the brain, adrenal gland and testis. ACSBG1 depletion did not significantly reduce the total ACS enzyme activity in these tissue types. In adult mouse brain, ACSBG1 expression was highest in the cerebellum; the low levels detected during the first week of life dramatically increased thereafter. Unexpectedly, lower, rather than higher, saturated VLCFA levels were found in cerebella from Acsbg1-/- vs. w.t. mice, especially after one week of age. Developmental changes in monounsaturated ω9 FA and polyunsaturated ω3 FA levels also differed between w.t. and Acsbg1-/- mice. ACSBG1 deficiency impacted the developmental expression of several cerebellar FA metabolism enzymes, including those required for the synthesis of ω3 polyunsaturated FA, precursors of bioactive signaling molecules like eicosanoids and docosanoids. These changes in membrane lipid FA composition likely affect membrane fluidity and may thus influence the body's response to inflammation. We conclude that, despite compelling circumstantial evidence, it is unlikely that ACSBG1 directly contributes to the pathology of XALD, decreasing its potential as a therapeutic target. Instead, the effects of ACSBG1 knockout on processes regulated by eicosanoids and/or docosanoids should be further investigated.

Adrenal insufficiency (AI) manifests as a clinical syndrome arising from either the direct impairment of adrenal glands, leading to primary AI characterized by deficiencies in glucocorticoids and mineralocorticoids, or adrenal cortex atrophy due to diminished adrenocorticotropic hormone (ACTH) stimulation, a consequence of hypothalamic and/or pituitary damage, resulting in secondary AI. The diagnosis of AI is based on clinical assessment and biochemical tests, including basal hormone level measurements and stimulation tests. In evaluating the results of laboratory tests, it is necessary to consider factors that may influence both pre-analytical and analytical phases, as well as the chosen methodology. Correct diagnosis of adrenal insufficiency and timely initiation of suitable replacement therapy are paramount. These steps are crucial not only for managing the condition but also to avert potentially life-threatening adrenal crises.

X-linked adrenoleukodystrophy (ALD) is a neurometabolic disorder caused by pathogenic variants in ABCD1 resulting very long-chain fatty acids (VLCFA) accumulation in plasma and tissues. Males can present with various clinical manifestations, including adrenal insufficiency, spinal cord disease, and leukodystrophy. Female patients typically develop spinal cord disease and peripheral neuropathy. Predicting the clinical outcome of an individual patient remains impossible due to the lack of genotype-phenotype correlation and predictive biomarkers.

The availability of a large prospective cohort of well-characterized patients and associated biobank samples allowed us to investigate the relationship between lipidome and disease severity in ALD. We performed a lipidomic analysis of plasma samples from 24 healthy controls, 92 male and 65 female ALD patients.

Here we show that VLCFA are incorporated into different lipid classes, including lysophosphatidylcholines, phosphatidylcholines, triglycerides, and sphingomyelins. Our results show a strong association between higher levels of VLCFA-containing lipids and the presence of leukodystrophy, adrenal insufficiency, and severe spinal cord disease in male ALD patients. In female ALD patients, VLCFA-lipid levels correlate with X-inactivation patterns in blood mononuclear cells, and higher levels are associated with more severe disease manifestations. Finally, hematopoietic stem cell transplantation significantly reduces, but does not normalize, plasma C26:0-lysophosphatidylcholine levels in male ALD patients. Our findings are supported by the concordance of C26:0-lysophosphatidylcholine and total VLCFA analysis with the lipidomics results.

This study reveals the profound impact of ALD on the lipidome and provides potential biomarkers for predicting clinical outcomes in ALD patients.

Human ABC transporters ABCD1-3 are all localized on the peroxisomal membrane and participate in the β-oxidation of fatty acyl-CoAs, but they differ from each other in substrate specificity. The transport of branched-chain fatty acids from cytosol to peroxisome is specifically driven by ABCD3, dysfunction of which causes severe liver diseases such as hepatosplenomegaly. Here we report two cryogenic electron microscopy (cryo-EM) structures of ABCD3 bound to phytanoyl-CoA and ATP at resolutions of 2.9 Å and 3.2 Å, respectively. A pair of phytanoyl-CoA molecules were observed in ABCD3, each binding to one transmembrane domain (TMD), which is distinct from our previously reported structure of ABCD1, where each fatty acyl-CoA molecule strongly crosslinks two TMDs. Upon ATP binding, ABCD3 exhibits a conformation that is open towards the peroxisomal matrix, leaving two extra densities corresponding to two CoA molecules deeply embedded in the translocation cavity. Structural analysis combined with substrate-stimulated ATPase activity assays indicated that the present structures might represent two states of ABCD3 in the transport cycle. These findings advance our understanding of fatty acid oxidation and the molecular pathology of related diseases.

X-linked adrenoleukodystrophy (ALD), an inherited neurometabolic disorder caused by mutations in ABCD1, which encodes the peroxisomal ABC transporter, mainly affects the brain, spinal cord, adrenal glands, and testes. In ALD patients, very-long-chain fatty acids (VLCFAs) fail to enter the peroxisome and undergo subsequent β-oxidation, resulting in their accumulation in the body. It has not been tested whether in vivo base editing or prime editing can be harnessed to ameliorate ALD. We developed a humanized mouse model of ALD by inserting a human cDNA containing the pathogenic variant into the mouse Abcd1 locus. The humanized ALD model showed increased levels of VLCFAs. To correct the mutation, we tested both base editing and prime editing and found that base editing using ABE8e(V106W) could correct the mutation in patient-derived fibroblasts at an efficiency of 7.4%. Adeno-associated virus (AAV)-mediated systemic delivery of NG-ABE8e(V106W) enabled robust correction of the pathogenic variant in the mouse brain (correction efficiency: ∼5.5%), spinal cord (∼5.1%), and adrenal gland (∼2%), leading to a significant reduction in the plasma levels of C26:0/C22:0. This established humanized mouse model and the successful correction of the pathogenic variant using a base editor serve as a significant step toward treating human ALD disease.

Background: 3,5,3'-Triiodothyroacetic acid (TRIAC) is a T3-receptor agonist pharmacologically used in patients to mitigate T3 resistance. It is additionally explored to treat some symptoms of patients with inactivating mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8, SLC16A2). MCT8 is expressed along the blood-brain barrier, on neurons, astrocytes, and oligodendrocytes. Hence, pathogenic variants in MCT8 limit the access of TH into and their functions within the brain. TRIAC was shown to enter the brain independently of MCT8 and to modulate expression of TH-dependent genes. The aim of the study was to identify transporters that facilitate TRIAC uptake into cells. Methods: We performed a whole-genome RNAi screen in HepG2 cells stably expressing a T3-receptor-dependent luciferase reporter gene. Validation of hits from the primary and confirmatory secondary screen involved a counter screen with siRNAs and compared the cellular response to TRIAC to the effect of T3, in order to exclude siRNAs targeting the gene expression machinery. MDCK1 cells were stably transfected with cDNA encoding C-terminally myc-tagged versions of the identified TRIAC-preferring transporters. Several individual clones were selected after immunocytochemical characterization for biochemical characterization of their 125I-TRIAC transport activities. Results: We identified SLC22A9 and SLC29A2 as transporters mediating cellular uptake of TRIAC. SLC22A9 encodes the organic anion transporter 7 (OAT7), a sodium-independent organic anion transporter expressed in the plasma membrane in brain, pituitary, liver, and other organs. Competition with the SLC22A9/OAT7 substrate estrone-3-sulfate reduced 125I-TRIAC uptake. SLC29A2 encodes the equilibrative nucleoside transporter 2 (ENT2), which is ubiquitously expressed, including pituitary and brain. Coincubation with the SLC29A2/ENT2 inhibitor nitrobenzyl-6-thioinosine reduced 125I-TRIAC uptake. Moreover, ABCD1, an ATP-dependent peroxisomal pump, was identified as a 125I-TRIAC exporter in transfected MDCK1 cells. Conclusions: Knowledge of TRIAC transporter expression patterns, also during brain development, may thus in the future help to interpret observations on TRIAC effects, as well as understand why TRIAC may not show a desirable effect on cells or organs not expressing appropriate transporters. The identification of ABCD1 highlights the sensitivity of our established screening assay, but it may not hold significant relevance for patients undergoing TRIAC treatment.

The "bubblegum" acyl-CoA synthetase (ACSBG1) is a pivotal player in lipid metabolism during the development of the mouse brain, facilitating the activation of long-chain fatty acids (LCFAs) and their integration into essential lipid species crucial for brain function. Through its enzymatic activity, ACSBG1 converts LCFAs into acyl-CoA derivatives, supporting vital processes like membrane formation, myelination, and energy production. Its regulatory role significantly influences neuronal growth, synaptic plasticity, and overall brain development, highlighting its importance in maintaining lipid homeostasis and proper brain function. Originally discovered in the fruit fly brain, ACSBG1 attracted attention for its potential implication in X-linked adrenoleukodystrophy (XALD) pathogenesis. Studies using Drosophila melanogaster lacking the ACSBG1 homolog, bubblegum, revealed adult neurodegeneration with elevated levels of very long-chain fatty acids (VLCFA). To explore ACSBG1's role in fatty acid (FA) metabolism and its relevance to XALD, we created an ACSBG1 knockout (Acsbg1-/-) mouse model and examined its impact on lipid metabolism during mouse brain development. Phenotypically, Acsbg1-/- mice resembled wild type (w.t.) mice. Despite its primary expression in tissues affected by XALD, brain, adrenal gland and testis, ACSBG1 depletion did not significantly reduce total ACS enzyme activity in these tissues when using LCFA or VLCFA as substrates. However, analysis unveiled intriguing developmental and compositional changes in FA levels associated with ACSBG1 deficiency. In the adult mouse brain, ACSBG1 expression peaked in the cerebellum, with lower levels observed in other brain regions. Developmentally, ACSBG1 expression in the cerebellum was initially low during the first week of life but increased dramatically thereafter. Cerebellar FA levels were assessed in both w.t. and Acsbg1-/- mouse brains throughout development, revealing notable differences. While saturated VLCFA levels were typically high in XALD tissues and in fruit flies lacking ACSBG1, cerebella from Acsbg1-/- mice displayed lower saturated VLCFA levels, especially after about 8 days of age. Additionally, monounsaturated ω9 FA levels exhibited a similar trend as saturated VLCFA, while ω3 polyunsaturated FA levels were elevated in Acsbg1-/- mice. Further analysis of specific FA levels provided additional insights into potential roles for ACSBG1. Notably, the decreased VLCFA levels in Acsbg1-/- mice primarily stemmed from changes in C24:0 and C26:0, while reduced ω9 FA levels were mainly observed in C18:1 and C24:1. ACSBG1 depletion had minimal effects on saturated long-chain FA or ω6 polyunsaturated FA levels but led to significant increases in specific ω3 FA, such as C20:5 and C22:5. Moreover, the impact of ACSBG1 deficiency on the developmental expression of several cerebellar FA metabolism enzymes, including those required for synthesis of ω3 polyunsaturated FA, was assessed; these FA can potentially be converted into bioactive signaling molecules like eicosanoids and docosanoids. In conclusion, despite compelling circumstantial evidence, it is unlikely that ACSBG1 directly contributes to the pathology of XALD. Instead, the effects of ACSBG1 knockout on processes regulated by eicosanoids and/or docosanoids should be further investigated.

The central nervous system (CNS) is the most delicate system in human body, with the most complex structure and function. It is vulnerable to trauma, infection, neurodegeneration and autoimmune diseases, and activates the immune system. An appropriate inflammatory response contributes to defence against invading microbes, whereas an excessive inflammatory response can aggravate tissue damage. The NLRP3 inflammasome was the first one studied in the brain. Once primed and activated, it completes the assembly of inflammasome (sensor NLRP3, adaptor ASC, and effector caspase-1), leading to caspase-1 activation and increased release of downstream inflammatory cytokines, as well as to pyroptosis. Cumulative studies have confirmed that NLRP3 plays an important role in regulating innate immunity and autoimmune diseases, and its inhibitors have shown good efficacy in animal models of various inflammatory diseases. In this review, we will briefly discuss the biological characteristics of NLRP3 inflammasome, summarize the recent advances and clinical impact of the NLRP3 inflammasome in infectious, inflammatory, immune, degenerative, genetic, and vascular diseases of CNS, and discuss the potential and challenges of NLRP3 as a therapeutic target for CNS diseases.

Peroxisomes are eukaryotic organelles that are essential for multiple metabolic pathways, including fatty acid oxidation, degradation of amino acids, and biosynthesis of ether lipids. Consequently, peroxisome dysfunction leads to pediatric-onset neurodegenerative conditions, including Peroxisome Biogenesis Disorders (PBD). Due to the dynamic, tissue-specific, and context-dependent nature of their biogenesis and function, live cell imaging of peroxisomes is essential for studying peroxisome regulation, as well as for the diagnosis of PBD-linked abnormalities. However, the peroxisomal imaging toolkit is lacking in many respects, with no reporters for substrate import, nor cell-permeable probes that could stain dysfunctional peroxisomes. Here we report that the BODIPY-C12 fluorescent fatty acid probe stains functional and dysfunctional peroxisomes in live mammalian cells. We then go on to improve BODIPY-C12, generating peroxisome-specific reagents, PeroxiSPY650 and PeroxiSPY555. These probes combine high peroxisome specificity, bright fluorescence in the red and far-red spectrum, and fast non-cytotoxic staining, making them ideal tools for live cell, whole organism, or tissue imaging of peroxisomes. Finally, we demonstrate that PeroxiSPY enables diagnosis of peroxisome abnormalities in the PBD CRISPR/Cas9 cell models and patient-derived cell lines.

ATP-binding cassette (ABC) transporters play a crucial role for the efflux of a wide range of substrates across different cellular membranes. In the central nervous system (CNS), ABC transporters have recently gathered significant attention due to their pivotal involvement in brain physiology and neurodegenerative disorders, such as Alzheimer's disease (AD). Glial cells are fundamental for normal CNS function and engage with several ABC transporters in different ways. Here, we specifically highlight ABC transporters involved in the maintenance of brain homeostasis and their implications in its metabolic regulation. We also show new aspects related to ABC transporter function found in less recognized diseases, such as Huntington's disease (HD) and experimental autoimmune encephalomyelitis (EAE), as a model for multiple sclerosis (MS). Understanding both their impact on the physiological regulation of the CNS and their roles in brain diseases holds promise for uncovering new therapeutic options. Further investigations and preclinical studies are warranted to elucidate the complex interplay between glial ABC transporters and physiological brain functions, potentially leading to effective therapeutic interventions also for rare CNS disorders.

Acyl-CoA binding domain containing 5 (ACBD5) is a critical player in handling very long chain fatty acids (VLCFA) en route for peroxisomal β-oxidation. Mutations in ACBD5 lead to the accumulation of VLCFA and patients present retinal dystrophy, ataxia, psychomotor delay and a severe leukodystrophy. Using CRISPR/Cas9, we generated and characterized an Acbd5 Gly357* mutant allele. Gly357* mutant mice recapitulated key features of the human disorder, including reduced survival, impaired locomotion and reflexes, loss of photoreceptors, and demyelination. The ataxic presentation of Gly357* mice involved the loss of cerebellar Purkinje cells and a giant axonopathy throughout the CNS. Lipidomic studies provided evidence for the extensive lipid dysregulation caused by VLCFA accumulation. Following a proteomic survey, functional studies in neurons treated with VLCFA unravelled a deregulated cytoskeleton with reduced actin dynamics and increased neuronal filopodia. We also show that an adeno-associated virus-mediated gene delivery ameliorated the gait phenotypes and the giant axonopathy, also improving myelination and astrocyte reactivity. Collectively, we established a mouse model with significance for VLCFA-related disorders. The development of relevant neuropathological outcomes enabled the understanding of mechanisms modulated by VLCFA and the evaluation of the efficacy of preclinical therapeutic interventions.

Very long-chain fatty acids (VLCFAs) are degraded exclusively in peroxisomes, as evidenced by the accumulation of VLCFAs in patients with certain peroxisomal disorders. Although accumulation of VLCFAs is considered to be associated with health issues, including neuronal degeneration, the mechanisms underlying VLCFAs-induced tissue degeneration remain unclear. Here, we report the toxic effect of VLCFA and protective effect of C18: 1 FA in peroxisome-deficient CHO cells. We examined the cytotoxicity of saturated and monounsaturated VLCFAs with chain-length at C20-C26, and found that longer and saturated VLCFA showed potent cytotoxicity at lower accumulation levels. Furthermore, the extent of VLCFA-induced toxicity was found to be associated with a decrease in cellular C18:1 FA levels. Notably, supplementation with C18:1 FA effectively rescued the cells from VLCFA-induced apoptosis without reducing the cellular VLCFAs levels, implying that peroxisome-deficient cells can survive in the presence of accumulated VLCFA, as long as the cells keep sufficient levels of cellular C18:1 FA. These results suggest a therapeutic potential of C18:1 FA in peroxisome disease and may provide new insights into the pharmacological effect of Lorenzo's oil, a 4:1 mixture of C18:1 and C22:1 FA.

Adrenoleukodystrophy (ALD) is a multifaceted, X-linked, neurodegenerative disorder that comprises several clinical phenotypes. ALD affects patients through a variety of physical, emotional, social, and other disease-specific factors that collectively contribute to disease burden. To facilitate clinical care and research, it is important to identify which symptoms are most common and relevant to individuals with any subtype of ALD.

We conducted semi-structured qualitative interviews and an international cross-sectional study to determine the most prevalent and important symptoms of ALD. Our study included adult participants with a diagnosis of ALD who were recruited from national and international patient registries. Responses were categorized by age, sex, disease phenotype, functional status, and other demographic and clinical features.

Seventeen individuals with ALD participated in qualitative interviews, providing 1709 direct quotes regarding their symptomatic burden. One hundred and nine individuals participated in the cross-sectional survey study, which inquired about 182 unique symptoms representing 24 distinct symptomatic themes. The symptomatic themes with the highest prevalence in the overall ALD sample cohort were problems with balance (90.9%), limitations with mobility or walking (87.3%), fatigue (86.4%), and leg weakness (86.4%). The symptomatic themes with the highest impact scores (on a 0-4 scale with 4 being the most severe) were trouble getting around (2.35), leg weakness (2.25), and problems with balance (2.21). A higher prevalence of symptomatic themes was associated with functional disability, employment disruption, and speech impairment.

There are many patient-relevant symptoms and themes that contribute to disease burden in individuals with ALD. These symptoms, identified by those having ALD, present key targets for further research and therapeutic development.

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.

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.

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.

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.

Kidney clear cell carcinoma (KIRC) is the most common subtype of renal cell carcinoma. Peroxisomes play a role in the regulation of tumorigenesis and cancer progression, yet the prognostic significance of peroxisome-related genes (PRGs) remains rarely studied. The study aimed to establish a novel prognostic risk model and identify potential biomarkers in KIRC.

The significant prognostic PRGs were screened through differential and Cox regression analyses, and LASSO Cox regression analysis was performed to establish a prognostic risk model in the training cohort, which was validated internally in the testing and entire cohorts, and further assessed in the GSE22541 cohort. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to explore the function and pathway differences between the high-risk and low-risk groups. The relationship between risk score and immune cell infiltration levels was evaluated in the CIBERSORT, ESTIMATE and TIMER databases. Finally, potential biomarkers were identified and validated from model genes, using immunohistochemistry.

Fourteen significant prognostic PRGs were identified using multiple analyses, and 9 genes (ABCD1, ACAD11, ACAT1, AGXT, DAO, EPHX2, FNDC5, HAO1, and HNGCLL1) were obtained to establish a prognostic model via LASSO Cox regression analysis. Combining the risk score with clinical factors to construct a nomogram, which provided support for personalized treatment protocols for KIRC patients. GO and KEGG analyses highlighted associations with substance metabolism, transport, and the PPAR signaling pathways. Tumor immune infiltration indicated immune suppression in the high-risk group, accompanied by higher tumor purity and the expression of 9 model genes was positively correlated with the level of immune cell infiltration. ACAT1 has superior prognostic capabilities in predicting the outcomes of KIRC patients.

The peroxisome-related prognostic risk model could better predict prognosis in KIRC patients.

Peroxisomal disorders can be classified as single-enzyme deficiencies or peroxisomal biogenesis disorders (characterized by multiple peroxisomal enzyme deficiencies or complete absence of peroxisomes). Most peroxisomal disorders give rise to complex multisystem disorders. Peroxisomal disorders associated with leukodystrophy are discussed in more detail, specifically X-linked adrenoleukodystrophy, Zellweger spectrum disorders, D-bifunctional protein deficiency, Acyl-CoA oxidase 1 deficiency, and Alpha-Methylacyl-CoA Racemase (AMACR) deficiency.

Inherited leukodystrophies are genetic disorders characterized by abnormal white matter in the central nervous system. Although individually rare, there are more than 400 distinct types of leukodystrophies with a cumulative incidence of 1 in 4500 live births. The pathophysiology of most leukodystrophies is poorly understood, there are treatments for only a few, and there is significant morbidity and mortality, suggesting a critical need for improvements in this field. A variety of animal, cell, and induced pluripotent stem cell-derived models have been developed for leukodystrophies, but with significant limitations in all models. Many leukodystrophies lack animal models, and extant models often show no or mixed recapitulation of key phenotypes. Zebrafish (Danio rerio) have become increasingly used as disease models for studying leukodystrophies due to their early onset of disease phenotypes and conservation of molecular and neurobiological mechanisms. Here, we focus on reviewing new zebrafish disease models for leukodystrophy or models with recent progress. This includes discussion of leukodystrophy with vanishing white matter disease, X-linked adrenoleukodystrophy, Zellweger spectrum disorders and peroxisomal disorders, PSAP deficiency, metachromatic leukodystrophy, Krabbe disease, hypomyelinating leukodystrophy-8/4H leukodystrophy, Aicardi-Goutières syndrome, RNASET2-deficient cystic leukoencephalopathy, hereditary diffuse leukoencephalopathy with spheroids-1 (CSF1R-related leukoencephalopathy), and ultra-rare leukodystrophies. Zebrafish models offer important potentials for the leukodystrophy field, including testing of new variants in known genes; establishing causation of newly discovered genes; and early lead compound identification for therapies. There are also unrealized opportunities to use humanized zebrafish models which have been sparsely explored.

Microglia are crucial for brain homeostasis, and dysfunction of these cells is a key driver in most neurodegenerative diseases, including peroxisomal leukodystrophies. In X-linked adrenoleukodystrophy (X-ALD), a neuroinflammatory disorder, very long-chain fatty acid (VLCFA) accumulation due to impaired degradation within peroxisomes results in microglial defects, but the underlying mechanisms remain unclear. Using CRISPR/Cas9 gene editing of key genes in peroxisomal VLCFA breakdown (Abcd1, Abcd2, and Acox1), we recently established easily accessible microglial BV-2 cell models to study the impact of dysfunctional peroxisomal β-oxidation and revealed a disease-associated microglial-like signature in these cell lines. Transcriptomic analysis suggested consequences on the immune response. To clarify how impaired lipid degradation impacts the immune function of microglia, we here used RNA-sequencing and functional assays related to the immune response to compare wild-type and mutant BV-2 cell lines under basal conditions and upon pro-inflammatory lipopolysaccharide (LPS) activation. A majority of genes encoding proinflammatory cytokines, as well as genes involved in phagocytosis, antigen presentation, and co-stimulation of T lymphocytes, were found differentially overexpressed. The transcriptomic alterations were reflected by altered phagocytic capacity, inflammasome activation, increased release of inflammatory cytokines, including TNF, and upregulated response of T lymphocytes primed by mutant BV-2 cells presenting peptides. Together, the present study shows that peroxisomal β-oxidation defects resulting in lipid alterations, including VLCFA accumulation, directly reprogram the main cellular functions of microglia. The elucidation of this link between lipid metabolism and the immune response of microglia will help to better understand the pathogenesis of peroxisomal leukodystrophies.

Adrenoleukodystrophy (ALD) is caused by pathogenic variants in the ABCD1 gene, encoding for the adrenoleukodystrophy protein (ALDP), leading to defective peroxisomal β-oxidation of very long-chain and branched-chain fatty acids (VLCFA). ALD manifests in both sexes with a spectrum of phenotypes, but approximately 35% of affected males develop childhood cerebral adrenoleukodystrophy (CCALD), which is lethal without hematopoietic stem cell transplant performed before symptoms start. Hence, ALD was added to the Recommended Uniform Screening Panel after the successful implementation in New York State (2013-2016). To date, thirty-five states have implemented newborn screening (NBS) for ALD, and a few programs have reported on the successes and challenges experienced. However, the overall impact of NBS on early detection of ALD has yet to be fully determined. Here, we conducted a retrospective analysis of VLCFA testing performed by our reference laboratory (ARUP Laboratories, Salt Lake City, UT, USA) over 10 years. Rate of detection, age at diagnosis, and male-to-female ratio were evaluated in patients with abnormal results before and after NBS implementation. After NBS inclusion, a significant increase in abnormal results was observed (471/6930, 6.8% vs. 384/11,670, 3.3%; p < 0.0001). Patients with ALDP deficiency identified via NBS were significantly younger (median age: 30 days vs. 21 years; p < 0.0001), and males and females were equally represented. ALD inclusion in NBS programs has increased pre-symptomatic detection of this disease, which is critical in preventing adrenal crisis as well as the severe cerebral form.

Leukodystrophies are a heterogenous group of inherited, degenerative encephalopathies, that if left untreated, are often lethal at an early age. Although some of the leukodystrophies can be treated with allogeneic hematopoietic stem cell transplantation, not all patients have suitable donors, and new treatment strategies, such as gene therapy, are rapidly being developed. Recent developments in the field of gene therapy for severe combined immune deficiencies, Leber's amaurosis, epidermolysis bullosa, Duchenne's muscular dystrophy and spinal muscular atrophy, have paved the way for the treatment of leukodystrophies, revealing some of the pitfalls, but overall showing promising results. Gene therapy offers the possibility for overexpression of secretable enzymes that can be released and through uptake, allow cross-correction of affected cells. Here, we discuss some of the leukodystrophies that have demonstrated strong potential for gene therapy interventions, such as X-linked adrenoleukodystrophy (X-ALD), and metachromatic leukodystrophy (MLD), which have reached clinical application. We further discuss the advantages and disadvantages of ex vivo lentiviral hematopoietic stem cell gene therapy, an approach for targeting microglia-like cells or rendering cross-correction. In addition, we summarize ongoing developments in the field of in vivo administration of recombinant adeno-associated viral (rAAV) vectors, which can be used for direct targeting of affected cells, and other recently developed molecular technologies that may be applicable to treating leukodystrophies in the future.

Fatty acid metabolism, including β-oxidation (βOX), plays an important role in human physiology and pathology. βOX is an essential process in the energy metabolism of most human cells. Moreover, βOX is also the source of acetyl-CoA, the substrate for (a) ketone bodies synthesis, (b) cholesterol synthesis, (c) phase II detoxication, (d) protein acetylation, and (d) the synthesis of many other compounds, including N-acetylglutamate-an important regulator of urea synthesis. This review describes the current knowledge on the importance of the mitochondrial and peroxisomal βOX in various organs, including the liver, heart, kidney, lung, gastrointestinal tract, peripheral white blood cells, and other cells. In addition, the diseases associated with a disturbance of fatty acid oxidation (FAO) in the liver, heart, kidney, lung, alimentary tract, and other organs or cells are presented. Special attention was paid to abnormalities of FAO in cancer cells and the diseases caused by mutations in gene-encoding enzymes involved in FAO. Finally, issues related to α- and ω- fatty acid oxidation are discussed.

X-linked adrenoleukodystrophy (X-ALD) is highly variable, ranging from slowly progressive adrenomyeloneuropathy to severe brain demyelination and inflammation (cerebral ALD, CALD) affecting males with childhood peak onset. Risk models integrating blood-based biomarkers to indicate CALD onset, enabling timely interventions, are lacking. Therefore, we evaluated the prognostic value of blood biomarkers in addition to current neuroimaging predictors for early detection of CALD.

We measured blood biomarkers in a retrospective, male CALD risk-assessment cohort consisting of 134 X-ALD patients and 66 controls and in a phenotype-blinded validation set (25 X-ALD boys, 4-13 years) using Simoa®and Luminex® technologies.

Among 25 biomarkers indicating axonal damage, astrocye/microglia activation, or immune-cell recruitment, neurofilament light chain (NfL) had the highest prognostic value for early indication of childhood/adolescent CALD. A plasma NfL cut-off level of 8.33 pg/mL, determined in the assessment cohort, correctly discriminated CALD with an accuracy of 96% [95% CI: 80-100] in the validation group. Multivariable logistic regression models revealed that combining NfL with GFAP or cytokines/chemokines (IL-15, IL-12p40, CXCL8, CCL11, CCL22, and IL-4) that were significantly elevated in CALD vs healthy controls had no additional benefit for detecting neuroinflammation. Some cytokines/chemokines were elevated only in childhood/adolescent CALD and already upregulated in asymptomatic X-ALD children (IL-15, IL-12p40, and CCL7). In adults, NfL levels distinguished CALD but were lower than in childhood/adolescent CALD patients with similar (MRI) lesion severity. Blood GFAP did not differentiate CALD from non-inflammatory X-ALD.

Biomarker-based risk prediction with a plasma NfL cut-off value of 8.33 pg/mL, determined by ROC analysis, indicates CALD onset with high sensitivity and specificity in childhood X-ALD patients. A specific pro-inflammatory cytokine/chemokine profile in asymptomatic X-ALD boys may indicate a primed, immanent inflammatory state aligning with peak onset of CALD. Age-related differences in biomarker levels in adult vs childhood CALD patients warrants caution in predicting onset and progression of CALD in adults. Further evaluations are needed to assess clinical utility of the NfL cut-off for risk prognosis of CALD onset.

Austrian Science Fund, European Leukodystrophy Association.

A number of hereditary ataxias are caused by inborn errors of metabolism (IEM), most of which are highly heterogeneous in their clinical presentation. Prompt diagnosis is important because disease-specific therapies may be available. In this review, we offer a comprehensive overview of metabolic ataxias summarized by disease, highlighting novel clinical trials and emerging therapies with a particular emphasis on first-in-human gene therapies. We present disease-specific treatments if they exist and review the current evidence for symptomatic treatments of these highly heterogeneous diseases (where cerebellar ataxia is part of their phenotype) that aim to improve the disease burden and enhance quality of life. In general, a multimodal and holistic approach to the treatment of cerebellar ataxia, irrespective of etiology, is necessary to offer the best medical care. Physical therapy and speech and occupational therapy are obligatory. Genetic counseling is essential for making informed decisions about family planning.

Contiguous ABCD1/ DXS1357E deletion syndrome (CADDS) is a rare deletion syndrome involving two contiguous genes on Xq28, ABCD1 and BCAP31 (formerly known as DXS1357E). Only nine individuals with this diagnosis have been reported in the medical literature to date. Intragenic loss-of-function variants in BCAP31 cause the deafness, dystonia, and cerebral hypomyelination syndrome (DDCH). Isolated pathogenic intragenic variants in ABCD1 are associated with the most common peroxisomal disorder, X-linked adrenoleukodystrophy (X-ALD), a single transporter deficiency, which in its more severe cerebral form is characterised by childhood-onset neurodegeneration and high levels of very-long-chain fatty acids (VLCFA). While increased VLCFA levels also feature in CADDS, the few patients described to date all presented as neonates with a severe phenotype. Here we report a tenth individual with CADDS, a male infant with dysmorphic facial features who was diagnosed through ultra-rapid whole genome sequencing (WGS) in the setting of persistent cholestatic liver disease, sensorineural hearing loss, hypotonia and growth failure and developmental delay. Biochemical studies showed elevated VLCFA and mildly reduced plasmalogens. He died at 7 months having developed pancreatic exocrine deficiency and interstitial lung disease, two features we propose to be possible extensions to the CADDS phenotype. We also review the genetic, phenotypic, and biochemical features in previously reported individuals with CADDS.

The advancements in population screening, including newborn screening, enables the identification of disease-causing variants and timely initiation of treatment. However, screening may also identify mild variants, non-disease variants, and variants of uncertain significance (VUS). The identification of a VUS poses a challenge in terms of diagnostic uncertainty and confusion. X-linked adrenoleukodystrophy (ALD) serves as an illustrative example of this complex issue. ALD is a monogenic neurometabolic disease with a complex clinical presentation and a lack of predictive tests for clinical severity. Despite the success of ALD newborn screening, a significant proportion (62%) of missense variants identified through newborn screening exhibit uncertainty regarding their pathogenicity. Resolving this issue requires ongoing efforts to accurately classify variants and refine screening protocols. While it is undisputable that ALD newborn screening greatly benefits boys with the disease, the identification of VUS underscores the need for continuous research and collaboration in improving screening practices.

Adrenoleukodystrophy (ALD) has a poor prognosis when it progresses to the cerebral form (CALD). The aim of this study is to investigate whether cerebrospinal fluid (CSF) neurofilament light chain (cNfL) is a sensitive biomarker for detecting CALD and assessing response to hematopoietic stem cell transplantation (HSCT).

We conducted a cross-sectional study of 41 male ALD patients. The cNfL levels in patients with the cerebral form of ALD (CALD) or the cerebello-brainstem form of ALD were compared with those in patients with adrenomyeloneuropathy (AMN). The correlation between cNfL levels and MRI-based Loes severity scores was investigated. A longitudinal analysis was performed on patients who underwent multiple CSF examinations.

The cNfL levels in 22 patients with CALD were significantly higher than those in 14 patients with AMN (median, 5545 vs. 1490 pg/mL; p < 0.001). The cutoff cNfL level of 1930 pg/mL showed good sensitivity (95.5%) and specificity (85.7%) for distinguishing CALD from AMN. The cNfL levels were positively correlated with Loes scores (p < 0.001). The cNfL levels in three AMN patients who later converted to CALD increased above the cutoff level during the conversion period, while the cNfL levels in four patients who remained in AMN were consistently below the cutoff. In 10 ALD patients who underwent HSCT, their cNfL levels decreased 3-24 months after HSCT. Two patients whose cNfL increased after HSCT showed deterioration in cognitive functions.

The cNfL level is useful for evaluating the disease activities of ALD and the response to HSCT.

X-linked adrenoleukodystrophy (X-ALD), the most frequent, inherited peroxisomal disease, is caused by mutations in the ABCD1 gene encoding a peroxisomal lipid transporter importing very long-chain fatty acids (VLCFAs) from the cytosol into peroxisomes for degradation via β-oxidation. ABCD1 deficiency results in accumulation of VLCFAs in tissues and body fluids of X-ALD patients with a wide range of phenotypic manifestations. The most severe variant, cerebral X-ALD (CALD) is characterized by progressive inflammation, loss of the myelin-producing oligodendrocytes and demyelination of the cerebral white matter. Whether the oligodendrocyte loss and demyelination in CALD are caused by a primary cell autonomous defect or injury to oligodendrocytes or by a secondary effect of the inflammatory reaction remains unresolved. To address the role of X-ALD oligodendrocytes in demyelinating pathophysiology, we combined the Abcd1 deficient X-ALD mouse model, in which VLCFAs accumulate without spontaneous demyelination, with the cuprizone model of toxic demyelination. In mice, the copper chelator cuprizone induces reproducible demyelination in the corpus callosum, followed by remyelination upon cuprizone removal. By immunohistochemical analyses of oligodendrocytes, myelin, axonal damage and microglia activation during de-and remyelination, we found that the mature oligodendrocytes of Abcd1 KO mice are more susceptible to cuprizone-induced cell death compared to WT mice in the early demyelinating phase. Furthermore, this effect was mirrored by a greater extent of acute axonal damage during demyelination in the KO mice. Abcd1 deficiency did not affect the function of microglia in either phase of the treatment. Also, the proliferation and differentiation of oligodendrocyte precursor cells and remyelination progressed at similar rates in both genotypes. Taken together, our findings point to an effect of Abcd1 deficiency on mature oligodendrocytes and the oligodendrocyte-axon unit, leading to increased vulnerability in the context of a demyelinating insult.

ABC transporters are essential for cellular physiology. Humans have 48 ABC genes organized into seven distinct families. Of these genes, 44 (in five distinct families) encode for membrane transporters, of which several are involved in drug resistance and disease pathways resulting from transporter dysfunction. Over the last decade, advances in structural biology have vastly expanded our mechanistic understanding of human ABC transporter function, revealing details of their molecular arrangement, regulation, and interactions, facilitated in large part by advances in cryo-EM that have rendered hitherto inaccessible targets amenable to high-resolution structural analysis. As a result, experimentally determined structures of multiple members of each of the five families of ABC transporters in humans are now available. Here we review this recent progress, highlighting the physiological relevance of human ABC transporters and mechanistic insights gleaned from their direct structure determination. We also discuss the impact and limitations of model systems and structure prediction methods in understanding human ABC transporters and discuss current challenges and future research directions.

Blood-brain barrier disruption marks the onset of cerebral adrenoleukodystrophy (CALD), a devastating cerebral demyelinating disease caused by loss of ABCD1 gene function. The underlying mechanism are not well understood, but evidence suggests that microvascular dysfunction is involved. We analyzed cerebral perfusion imaging in boys with CALD treated with autologous hematopoietic stem-cells transduced with the Lenti-D lentiviral vector that contains ABCD1 cDNA as part of a single group, open-label phase 2-3 safety and efficacy study (NCT01896102) and patients treated with allogeneic hematopoietic stem cell transplantation. We found widespread and sustained normalization of white matter permeability and microvascular flow. We demonstrate that ABCD1 functional bone marrow-derived cells can engraft in the cerebral vascular and perivascular space. Inverse correlation between gene dosage and lesion growth suggests that corrected cells contribute long-term to remodeling of brain microvascular function. Further studies are needed to explore the longevity of these effects.