Leriglitazone halts disease progression in adult patients with early cerebral adrenoleukodystrophy.

Cerebral adrenoleukodystrophy (CALD) is an X-linked rapidly progressive demyelinating disease leading to death usually within a few years. The standard of care is hematopoietic stem cell transplantation (HSCT), but many men are not eligible due to age, absence of a matched donor, or lesions of the corticospinal tracts (CST). Based on the ADVANCE study showing that leriglitazone decreases the occurrence of CALD, we treated 13 adult CALD patients (19-67 years of age) either not eligible to HSCT (n= 8) or awaiting HSCT (n= 5). Patients were monitored every 3 months with standardized neurological scores, plasma biomarkers and brain MRI comprising lesion volumetrics and diffusion tensor imaging. The disease stabilized clinically and radiologically in 10 patients with up to 2 years of follow-up. Five patients presented with gadolinium enhancing CST lesions that all turned gadolinium negative and, remarkably, regressed in four patients. Plasma neurofilament light chain levels stabilized in all 10 patients and correlated with lesion load. The two patients who continued to deteriorate were over 60 years of age with prominent cognitive impairment. One patient rapidly died from Covid19. These results suggest that leriglitazone can arrest disease progression in adults with early-stage CALD and may be an alternative treatment to HSCT.

Saturated very long-chain fatty acids regulate macrophage plasticity and invasiveness.

Saturated very long-chain fatty acids (VLCFA, ≥ C22), enriched in brain myelin and innate immune cells, accumulate in X-linked adrenoleukodystrophy (X-ALD) due to inherited dysfunction of the peroxisomal VLCFA transporter ABCD1. In its severest form, X-ALD causes cerebral myelin destruction with infiltration of pro-inflammatory skewed monocytes/macrophages. How VLCFA levels relate to macrophage activation is unclear. Here, whole transcriptome sequencing of X-ALD macrophages indicated that VLCFAs prime human macrophage membranes for inflammation and increased expression of factors involved in chemotaxis and invasion. When added externally to mimic lipid release in demyelinating X-ALD lesions, VLCFAs did not activate toll-like receptors in primary macrophages. In contrast, VLCFAs provoked pro-inflammatory responses through scavenger receptor CD36-mediated uptake, cumulating in JNK signalling and expression of matrix-degrading enzymes and chemokine release. Following pro-inflammatory LPS activation, VLCFA levels increased also in healthy macrophages. With the onset of the resolution, VLCFAs were rapidly cleared in control macrophages by increased peroxisomal VLCFA degradation through liver-X-receptor mediated upregulation of ABCD1. ABCD1 deficiency impaired VLCFA homeostasis and prolonged pro-inflammatory gene expression upon LPS treatment. Our study uncovers a pivotal role for ABCD1, a protein linked to neuroinflammation, and associated peroxisomal VLCFA degradation in regulating macrophage plasticity.

Impaired plasticity of macrophages in X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy is caused by ATP-binding cassette transporter D1 (ABCD1) mutations and manifests by default as slowly progressive spinal cord axonopathy with associated demyelination (adrenomyloneuropathy). In 60% of male cases, however, X-linked adrenoleukodystrophy converts to devastating cerebral inflammation and demyelination (cerebral adrenoleukodystrophy) with infiltrating blood-derived monocytes and macrophages and cytotoxic T cells that can only be stopped by allogeneic haematopoietic stem cell transplantation or gene therapy at an early stage of the disease. Recently, we identified monocytes/macrophages but not T cells to be severely affected metabolically by ABCD1 deficiency. Here we found by whole transcriptome analysis that, although monocytes of patients with X-linked adrenoleukodystrophy have normal capacity for macrophage differentiation and phagocytosis, they are pro-inflammatory skewed also in patients with adrenomyloneuropathy in the absence of cerebral inflammation. Following lipopolysaccharide activation, the ingestion of myelin debris, normally triggering anti-inflammatory polarization, did not fully reverse the pro-inflammatory status of X-linked adrenoleukodystrophy macrophages. Immunohistochemistry on post-mortem cerebral adrenoleukodystrophy lesions reflected the activation pattern by prominent presence of enlarged lipid-laden macrophages strongly positive for the pro-inflammatory marker co-stimulatory molecule CD86. Comparative analyses of lesions with matching macrophage density in cases of cerebral adrenoleukodystrophy and acute multiple sclerosis showed a similar extent of pro-inflammatory activation but a striking reduction of anti-inflammatory mannose receptor (CD206) and haemoglobin-haptoglobin receptor (CD163) expression on cerebral adrenoleukodystrophy macrophages. Accordingly, ABCD1-deficiency leads to an impaired plasticity of macrophages that is reflected in incomplete establishment of anti-inflammatory responses, thus possibly contributing to the devastating rapidly progressive demyelination in cerebral adrenoleukodystrophy that only in rare cases arrests spontaneously. These findings emphasize monocytes/macrophages as crucial therapeutic targets for preventing or stopping myelin destruction in patients with X-linked adrenoleukodystrophy.

LXR antagonists induce ABCD2 expression.

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

Involvement of human peroxisomes in biosynthesis and signaling of steroid and peptide hormones.

Although peroxisomes exert essential biological functions, cell type-specific features of this important organelle are still only superficially characterized. An intriguing new aspect of peroxisomal function was recently uncovered by the observation that the peptide hormones ß-lipotropin (ß-LPH) and ß-endorphin are localized to peroxisomes in various human tissues. This suggests a functional link between peptide hormone metabolism and peroxisomes. In addition, because endocrine manifestations that affect steroid hormones are often found in patients suffering from inherited peroxisomal disorders, the question has been raised whether peroxisomes are also involved in steroidogenesis. With this chapter, we will review several crucial aspects concerning peroxisomes and hormone metabolism.

High-resolution analysis of parent-of-origin allelic expression in the Arabidopsis Endosperm.

Genomic imprinting is an epigenetic phenomenon leading to parent-of-origin specific differential expression of maternally and paternally inherited alleles. In plants, genomic imprinting has mainly been observed in the endosperm, an ephemeral triploid tissue derived after fertilization of the diploid central cell with a haploid sperm cell. In an effort to identify novel imprinted genes in Arabidopsis thaliana, we generated deep sequencing RNA profiles of F1 hybrid seeds derived after reciprocal crosses of Arabidopsis Col-0 and Bur-0 accessions. Using polymorphic sites to quantify allele-specific expression levels, we could identify more than 60 genes with potential parent-of-origin specific expression. By analyzing the distribution of DNA methylation and epigenetic marks established by Polycomb group (PcG) proteins using publicly available datasets, we suggest that for maternally expressed genes (MEGs) repression of the paternally inherited alleles largely depends on DNA methylation or PcG-mediated repression, whereas repression of the maternal alleles of paternally expressed genes (PEGs) predominantly depends on PcG proteins. While maternal alleles of MEGs are also targeted by PcG proteins, such targeting does not cause complete repression. Candidate MEGs and PEGs are enriched for cis-proximal transposons, suggesting that transposons might be a driving force for the evolution of imprinted genes in Arabidopsis. In addition, we find that MEGs and PEGs are significantly faster evolving when compared to other genes in the genome. In contrast to the predominant location of mammalian imprinted genes in clusters, cluster formation was only detected for few MEGs and PEGs, suggesting that clustering is not a major requirement for imprinted gene regulation in Arabidopsis.

Peroxisomal Localization of a Truncated HMG-CoA Reductase under Low Cholesterol Conditions.

3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase, HMGCR) is one of the rate-limiting enzymes in the mevalonate pathway required for cholesterol biosynthesis. It is an integral membrane protein of the endoplasmic reticulum (ER) but has occasionally been described in peroxisomes. By co-immunofluorescence microscopy using different HMGCR antibodies, we present evidence for a dual localization of HMGCR in the ER and peroxisomes in differentiated human monocytic THP-1 cells, primary human monocyte-derived macrophages and human primary skin fibroblasts under conditions of low cholesterol and statin treatment. Using density gradient centrifugation and Western blot analysis, we observed a truncated HMGCR variant of 76 kDa in the peroxisomal fractions, while a full-length HMGCR of 96 kDa was contained in fractions of the ER. In contrast to primary human control fibroblasts, peroxisomal HMGCR was not found in fibroblasts from patients suffering from type-1 rhizomelic chondrodysplasia punctata, who lack functional PEX7 and, thus, cannot import peroxisomal matrix proteins harboring a type-2 peroxisomal targeting signal (PTS2). Moreover, in the N-terminal region of the soluble 76 kDa C-terminal catalytic domain, we identified a PTS2-like motif, which was functional in a reporter context. We propose that under sterol-depleted conditions, part of the soluble HMGCR domain, which is released from the ER by proteolytic processing for further turnover, remains sufficiently long in the cytosol for peroxisomal import via a PTS2/PEX7-dependent mechanism. Altogether, our findings describe a dual localization of HMGCR under combined lipid depletion and statin treatment, adding another puzzle piece to the complex regulation of HMGCR.

H3K27me3 profiling of the endosperm implies exclusion of polycomb group protein targeting by DNA methylation.

Polycomb group (PcG) proteins act as evolutionary conserved epigenetic mediators of cell identity because they repress transcriptional programs that are not required at particular developmental stages. Each tissue is likely to have a specific epigenetic profile, which acts as a blueprint for its developmental fate. A hallmark for Polycomb Repressive Complex 2 (PRC2) activity is trimethylated lysine 27 on histone H3 (H3K27me3). In plants, there are distinct PRC2 complexes for vegetative and reproductive development, and it was unknown so far whether these complexes have target gene specificity. The Fertilization Independent Seed (FIS) PRC2 complex is specifically expressed in the endosperm and is required for its development; loss of FIS function causes endosperm hyperproliferation and seed abortion. The endosperm nourishes the embryo, similar to the physiological function of the placenta in mammals. We established the endosperm H3K27me3 profile and identified specific target genes of the FIS complex with functional roles in endosperm cellularization and chromatin architecture, implicating that distinct PRC2 complexes have a subset of specific target genes. Importantly, our study revealed that selected transposable elements and protein coding genes are specifically targeted by the FIS PcG complex in the endosperm, whereas these elements and genes are densely marked by DNA methylation in vegetative tissues, suggesting that DNA methylation prevents targeting by PcG proteins in vegetative tissues.

Putative Arabidopsis transcriptional adaptor protein (PROPORZ1) is required to modulate histone acetylation in response to auxin.

Plant development is highly adaptable and controlled by a combination of various regulatory circuits that integrate internal and environmental cues. The phytohormone auxin mediates such growth responses, acting as a dynamic signal in the control of morphogenesis via coordinating the interplay between cell cycle progression and cell differentiation. Mutants in the chromatin-remodeling component PROPORZ1 (PRZ1; also known as AtADA2b) are impaired in auxin effects on morphogenesis, suggestive of an involvement of PRZ1-dependent control of chromatin architecture in the determination of hormone responses. Here we demonstrate that PRZ1 is required for accurate histone acetylation at auxin-controlled loci. Specifically, PRZ1 is involved in the modulation of histone modifications and corresponding adjustments in gene expression of Arabidopsis KIP RELATED PROTEIN (KRP) CDK inhibitor genes in response to auxin. Deregulated KRP expression in KRP silencer lines phenocopies prz1 hyperproliferative growth phenotypes, whereas in a KRP overexpression background some mutant phenotypes are suppressed. Collectively, our findings support a model in which translation of positional signals into developmental cues involves adjustments in chromatin modifications that orchestrate auxin effects on cell proliferation.

ABCD1 Transporter Deficiency Results in Altered Cholesterol Homeostasis.

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

Efficacy of HDAC Inhibitors in Driving Peroxisomal ß-Oxidation and Immune Responses in Human Macrophages: Implications for Neuroinflammatory Disorders.

Elevated levels of saturated very long-chain fatty acids (VLCFAs) in cell membranes and secreted lipoparticles have been associated with neurotoxicity and, therefore, require tight regulation. Excessive VLCFAs are imported into peroxisomes for degradation by ß-oxidation. Impaired VLCFA catabolism due to primary or secondary peroxisomal alterations is featured in neurodegenerative and neuroinflammatory disorders such as X-linked adrenoleukodystrophy and multiple sclerosis (MS). Here, we identified that healthy human macrophages upregulate the peroxisomal genes involved in ß-oxidation during myelin phagocytosis and pro-inflammatory activation, and that this response is impaired in peripheral macrophages and phagocytes in brain white matter lesions in MS patients. The pharmacological targeting of VLCFA metabolism and peroxisomes in innate immune cells could be favorable in the context of neuroinflammation and neurodegeneration. We previously identified the epigenetic histone deacetylase (HDAC) inhibitors entinostat and vorinostat to enhance VLCFA degradation and pro-regenerative macrophage polarization. However, adverse side effects currently limit their use in chronic neuroinflammation. Here, we focused on tefinostat, a monocyte/macrophage-selective HDAC inhibitor that has shown reduced toxicity in clinical trials. By using a gene expression analysis, peroxisomal ß-oxidation assay, and live imaging of primary human macrophages, we assessed the efficacy of tefinostat in modulating VLCFA metabolism, phagocytosis, chemotaxis, and immune function. Our results revealed the significant stimulation of VLCFA degradation with the upregulation of genes involved in peroxisomal ß-oxidation and interference with immune cell recruitment; however, tefinostat was less potent than the class I HDAC-selective inhibitor entinostat in promoting a regenerative macrophage phenotype. Further research is needed to fully explore the potential of class I HDAC inhibition and downstream targets in the context of neuroinflammation.

Immune response of BV-2 microglial cells is impacted by peroxisomal beta-oxidation.

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 ß-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 ß-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.

Biomarker-based risk prediction for the onset of neuroinflammation in X-linked adrenoleukodystrophy.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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. INTERPRETATION: 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. FUNDING: Austrian Science Fund, European Leukodystrophy Association.

Peroxisomal very long-chain fatty acid transport is targeted by herpesviruses and the antiviral host response.

Very long-chain fatty acids (VLCFA) are critical for human cytomegalovirus replication and accumulate upon infection. Here, we used Epstein-Barr virus (EBV) infection of human B cells to elucidate how herpesviruses target VLCFA metabolism. Gene expression profiling revealed that, despite a general induction of peroxisome-related genes, EBV early infection decreased expression of the peroxisomal VLCFA transporters ABCD1 and ABCD2, thus impairing VLCFA degradation. The mechanism underlying ABCD1 and ABCD2 repression involved RNA interference by the EBV-induced microRNAs miR-9-5p and miR-155, respectively, causing significantly increased VLCFA levels. Treatment with 25-hydroxycholesterol, an antiviral innate immune modulator produced by macrophages, restored ABCD1 expression and reduced VLCFA accumulation in EBV-infected B-lymphocytes, and, upon lytic reactivation, reduced virus production in control but not ABCD1-deficient cells. Finally, also other herpesviruses and coronaviruses target ABCD1 expression. Because viral infection might trigger neuroinflammation in X-linked adrenoleukodystrophy (X-ALD, inherited ABCD1 deficiency), we explored a possible link between EBV infection and cerebral X-ALD. However, neither immunohistochemistry of post-mortem brains nor analysis of EBV seropositivity in 35 X-ALD children supported involvement of EBV in the onset of neuroinflammation. Collectively, our findings indicate a previously unrecognized, pivotal role of ABCD1 in viral infection and host defence, prompting consideration of other viral triggers in cerebral X-ALD.

The brain penetrant PPARγ agonist leriglitazone restores multiple altered pathways in models of X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD), a potentially fatal neurometabolic disorder with no effective pharmacological treatment, is characterized by clinical manifestations ranging from progressive spinal cord axonopathy [adrenomyeloneuropathy (AMN)] to severe demyelination and neuroinflammation (cerebral ALD-cALD), for which molecular mechanisms are not well known. Leriglitazone is a recently developed brain penetrant full PPARγ agonist that could modulate multiple biological pathways relevant for neuroinflammatory and neurodegenerative diseases, and particularly for X-ALD. We found that leriglitazone decreased oxidative stress, increased adenosine 5'-triphosphate concentration, and exerted neuroprotective effects in primary rodent neurons and astrocytes after very long chain fatty acid-induced toxicity simulating X-ALD. In addition, leriglitazone improved motor function; restored markers of oxidative stress, mitochondrial function, and inflammation in spinal cord tissues from AMN mouse models; and decreased the neurological disability in the EAE neuroinflammatory mouse model. X-ALD monocyte-derived patient macrophages treated with leriglitazone were less skewed toward an inflammatory phenotype, and the adhesion of human X-ALD monocytes to brain endothelial cells decreased after treatment, suggesting the potential of leriglitazone to prevent the progression to pathologically disrupted blood-brain barrier. Leriglitazone increased myelin debris clearance in vitro and increased myelination and oligodendrocyte survival in demyelination-remyelination in vivo models, thus promoting remyelination. Last, leriglitazone was clinically tested in a phase 1 study showing central nervous system target engagement (adiponectin increase) and changes on inflammatory biomarkers in plasma and cerebrospinal fluid. The results of our study support the use of leriglitazone in X-ALD and, more generally, in other neuroinflammatory and neurodegenerative conditions.

Neurofilament light chain as a potential biomarker for monitoring neurodegeneration in X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD), the most frequent monogenetic disorder of brain white matter, is highly variable, ranging from slowly progressive adrenomyeloneuropathy (AMN) to life-threatening inflammatory brain demyelination (CALD). In this study involving 94 X-ALD patients and 55 controls, we tested whether plasma/serum neurofilament light chain protein (NfL) constitutes an early distinguishing biomarker. In AMN, we found moderately elevated NfL with increased levels reflecting higher grading of myelopathy-related disability. Intriguingly, NfL was a significant predictor to discriminate non-converting AMN from cohorts later developing CALD. In CALD, markedly amplified NfL levels reflected brain lesion severity. In rare cases, atypically low NfL revealed a previously unrecognized smoldering CALD disease course with slowly progressive myelin destruction. Upon halt of brain demyelination by hematopoietic stem cell transplantation, NfL gradually normalized. Together, our study reveals that blood NfL reflects inflammatory activity and progression in CALD patients, thus constituting a potential surrogate biomarker that may facilitate clinical decisions and therapeutic development.

Targeting foam cell formation in inflammatory brain diseases by the histone modifier MS-275.

OBJECTIVE: To assess class I-histone deacetylase (HDAC) inhibition on formation of lipid-accumulating, disease-promoting phagocytes upon myelin load in vitro, relevant for neuroinflammatory disorders like multiple sclerosis (MS) and cerebral X-linked adrenoleukodystrophy (X-ALD). METHODS: Immunohistochemistry on postmortem brain tissue of acute MS (n = 6) and cerebral ALD (n = 4) cases to analyze activation and foam cell state of phagocytes. RNA-Seq of in vitro differentiated healthy macrophages (n = 8) after sustained myelin-loading to assess the metabolic shift associated with foam cell formation. RNA-Seq analysis of genes linked to lipid degradation and export in MS-275-treated human HAP1 cells and RT-qPCR analysis of HAP1 cells knocked out for individual members of class I HDACs or the corresponding enzymatically inactive knock-in mutants. Investigation of intracellular lipid/myelin content after MS-275 treatment of myelin-laden human foam cells. Analysis of disease characteristic very long-chain fatty acid (VLCFA) metabolism and inflammatory state in MS-275-treated X-ALD macrophages. RESULTS: Enlarged foam cells coincided with a pro-inflammatory, lesion-promoting phenotype in postmortem tissue of MS and cerebral ALD patients. Healthy in vitro myelin laden foam cells upregulated genes linked to LXRα/PPARγ pathways and mimicked a program associated with tissue repair. Treating these cells with MS-275, amplified this gene transcription program and significantly reduced lipid and cholesterol accumulation and, thus, foam cell formation. In macrophages derived from X-ALD patients, MS-275 improved the disease-associated alterations of VLCFA metabolism and reduced the pro-inflammatory status of these cells. INTERPRETATION: These findings identify class I-HDAC inhibition as a potential novel strategy to prevent disease promoting foam cell formation in CNS inflammation.

Vorinostat in the acute neuroinflammatory form of X-linked adrenoleukodystrophy.

OBJECTIVE: To identify a pharmacological compound targeting macrophages, the most affected immune cells in inflammatory X-linked adrenoleukodystrophy (cerebral X-ALD) caused by ABCD1 mutations and involved in the success of hematopoietic stem cell transplantation and gene therapy. METHODS: A comparative database analysis elucidated the epigenetic repressing mechanism of the related ABCD2 gene in macrophages and identified the histone deacetylase (HDAC) inhibitor Vorinostat as a compound to induce ABCD2 in these cells to compensate for ABCD1 deficiency. In these cells, we investigated ABCD2 and pro-inflammatory gene expression, restoration of defective peroxisomal ß-oxidation activity, accumulation of very long-chain fatty acids (VLCFAs) and their differentiation status. We investigated ABCD2 and pro-inflammatory gene expression, restoration of defective peroxisomal ß-oxidation activity, accumulation of very long-chain fatty acids (VLCFA) and differentiation status. Three advanced cerebral X-ALD patients received Vorinostat and CSF and MRI diagnostics was carried out in one patient after 80 days of treatment. RESULTS: Vorinostat improved the metabolic defects in X-ALD macrophages by stimulating ABCD2 expression, peroxisomal ß-oxidation, and ameliorating VLCFA accumulation. Vorinostat interfered with pro-inflammatory skewing of X-ALD macrophages by correcting IL12B expression and further reducing monocyte differentiation. Vorinostat normalized the albumin and immunoglobulin CSF-serum ratios, but not gadolinium enhancement upon 80 days of treatment. INTERPRETATION: The beneficial effects of HDAC inhibitors on macrophages in X-ALD and the improvement of the blood-CSF/blood-brain barrier are encouraging for future investigations. In contrast with Vorinostat, less toxic macrophage-specific HDAC inhibitors might improve also the clinical state of X-ALD patients with advanced inflammatory demyelination.

Distinct modulatory roles for thyroid hormone receptors TRalpha and TRbeta in SREBP1-activated ABCD2 expression.

Adrenoleukodystrophy-related protein, a peroxisomal ABC transporter encoded by ABCD2, displays functional redundancy with the disease-associated X-linked adrenoleukodystrophy protein, making pharmacological induction of ABCD2 a potentially attractive therapeutic approach. Sterol regulatory element (SRE)-binding proteins (SREBPs) induce ABCD2 through an SRE overlapping with a direct repeat (DR-4) element. Here we show that thyroid hormone (T(3)) receptor (TR)alpha and TRbeta bind this motif thereby modulating SREBP1-dependent activation of ABCD2. Unliganded TRbeta, but not TRalpha, represses ABCD2 induction independently of DNA binding. However, activation by TRalpha and derepression of TRbeta are T(3)-dependent and require intact SRE/DR-4 motifs. Electrophoretic mobility shift assays with nuclear extracts support a direct interaction of TR and SREBP1 at the SRE/DR-4. In the liver, Abcd2 expression is high in young mice (with high T(3) and TRalpha levels) but downregulated in adults (with low T(3) and TRalpha but elevated TRbeta levels). This temporal repression of Abcd2 is blunted in TRbeta-deficient mice, and the response to manipulated T(3) states is abrogated in TRalpha-deficient mice. These findings show that TRalpha and TRbeta differentially modulate SREBP1-activated ABCD2 expression at overlapping SRE/DR-4 elements, suggesting a novel mode of cross-talk between TR and SREBP in gene regulation.