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.

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.

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.

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.