Isabelle Weinhofer

Background The neuroinflammatory cerebral form of X-linked adrenoleukodystrophy (CALD) is among the most severe neurological diseases affecting children. Early intervention by hematopoietic stem cell transplantation (HSCT) or gene therapy halts CALD, justifying its inclusion in newborn screening programmes. Currently, eligibility for treatment is assessed using MRI-based Loes and neurological scoring; however, grading poorly differentiates advanced stages or atypical lesion patterns. We recently identified blood neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP), indicative of axonal and glial damage, as valid biomarkers reflecting CALD activity. Methods Here, we investigated how pre-treatment biomarker levels relate to outcome of HSCT or gene therapy in a retrospective cohort of 14 paediatric CALD patients aged 5–13 years. Patients were monitored longitudinally before and for up to 5.5 years following HSCT or gene therapy. Disease progression was assessed using MRI-based Loes scoring and neurological evaluations. Plasma NfL and GFAP were quantified using single molecule array (Simoa) technology. Results Here we show that blood NfL and GFAP transiently increase due to myeloablative conditioning and gradually normalize post-treatment. Pre-treatment NfL ≤ 113 pg/ml, but not GFAP, correlate with CALD stabilization or, for Loes >9, only minor progression. In contrast, NfL >243 pg/ml associates with major progression irrespective of baseline Loes score. In three boys with atypical lesions or advanced disease, NfL outperforms Loes-scoring in predicting outcome. Conclusions Blood NfL can complement clinical decision-making, particularly in patients with advanced CALD or atypical lesions. These findings are especially relevant for clinical management in countries that have not yet implemented X-linked adrenoleukodystrophy into newborn screening.

Understanding pathological changes in the brain is essential for guiding treatment decisions in brain injuries and diseases. Despite significant advances in brain imaging techniques, clinical practice still faces challenges due to infrastructure reliance and high resource demands. This review explores the current knowledge on blood‐based biomarkers that indicate brain pathology, which can assist in identifying at‐risk patients, diagnosing patients, predicting disease progression, and treatment response. We focus on the inherited metabolic disorders X‐linked adrenoleukodystrophy (X‐ALD) and metachromatic leukodystrophy (MLD) which share remarkable phenotypic variability. Disease‐specific increases in the lipid metabolites lyso‐PC26:0 in X‐ALD and sulfatides in MLD might contribute to predicting clinical manifestation. Disease‐unspecific biomarkers for axonal damage (neurofilament light chain protein, NfL) and glial degeneration (glial fibrillary acidic protein, GFAP) are able to distinguish X‐ALD and MLD phenotypes at the group level. The implementation of X‐ALD into newborn screening programs in various countries, including several U.S. states, has increased the demand for predictive blood‐based biomarkers capable of detecting the early conversion from the pre‐symptomatic to the early neuroinflammatory cerebral form of X‐ALD. Among different biomarkers, NfL has proven most effective in reflecting neuroinflammation and correlating with brain lesion volume and the magnetic resonance imaging (MRI)‐based severity scores. We discuss how NfL has moved from initial proof‐of‐principle towards proof‐of‐concept studies in brain disorders such as multiple sclerosis and how this knowledge could be applied for the clinical implementation of NfL in rare inherited metabolic disorders such as X‐ALD.

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.

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 β-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 β-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 β-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 β-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.

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.

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.

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.

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.