CRISPR/Cas9-mediated knockout of Abcd1 and Abcd2 genes in BV-2 cells: novel microglial models for X-linked Adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD), the most frequent peroxisomal disorder, is associated with mutation in the ABCD1 gene which encodes a peroxisomal ATP-binding cassette transporter for very long-chain fatty acids (VLCFA). The biochemical hallmark of the disease is the accumulation of VLCFA. Peroxisomal defect in microglia being now considered a priming event in the pathology, we have therefore generated murine microglial cells mutated in the Abcd1 gene and its closest homolog, the Abcd2 gene. Using CRISPR/Cas9 gene editing strategy, we obtained 3 cell clones with a single or double deficiency. As expected, only the combined absence of ABCD1 and ABCD2 proteins resulted in the accumulation of VLCFA. Ultrastructural analysis by electron microscopy revealed in the double mutant cells the presence of lipid inclusions similar to those observed in brain macrophages of patients. These observations are likely related to the increased level of cholesterol and the accumulation of neutral lipids that we noticed in mutant cells. A preliminary characterization of the impact of peroxisomal defects on the expression of key microglial genes such as Trem2 suggests profound changes in microglial functions related to inflammation and phagocytosis. The expression levels of presumed modifier genes have also been found modified in mutant cells, making these novel cell lines relevant for use as in vitro models to better understand the physiopathogenesis of X-ALD and to discover new therapeutic targets.     

Peroxisomal and mitochondrial status of two murine oligodendrocytic cell lines (158N, 158JP): potential models for the study of peroxisomal disorders associated with dysmyelination processes

In some neurodegenerative disorders (leukodystrophies) characterized by myelin alterations, the defect of peroxisomal functions on myelin-producing cells (oligodendrocytes) are poorly understood. The development of in vitro models is fundamental to understanding the physiopathogenesis of these diseases. We characterized two immortalized murine oligodendrocyte cell lines: a normal (158N) and a jimpy (158JP) cell line mutated for the proteolipid protein PLP/DM20. Fluorescence microscopy, flow cytometry, and western blotting analysis allow to identify major myelin proteins (PLP colocalizing with mitochondria; myelin basic protein), oligodendrocyte (CNPase and myelin oligodendrocyte glycoprotein), and peroxisomal markers [adrenoleukodystrophy protein, PMP70, acyl-CoA oxidase 1 (ACOX1), l -peroxisomal bifunctional enzyme, and catalase]. Using electron microscopy, peroxisomes were identified in the two cell lines. Gene expression (ATP-binding cassette, Abcd1 , Abcd2 , Abcd3 , and Acox1 ) involved in peroxisomal transport or β-oxidation of fatty acids was evaluated using quantitative PCR. 4-phenylbutyrate treatment increases expression of ACOX1, l -peroxisomal bifunctional enzyme, PLP, myelin oligodendrocyte glycoprotein, and CNPase, mainly in 158N cells. In both cell lines, 4-phenylbutyrate-induced ACOX1 and catalase activities while only Abcd2 gene was up-regulated in 158JP. Moreover, the higher mitochondrial activity and content observed in 158JP were associated with higher glutathione content and increased basal production of reactive oxygen species revealing different redox statuses. Altogether, 158N and 158JP cells will permit studying the relationships between peroxisomal defects, mitochondrial activity, and oligodendrocyte functions.     

Abcd2 Is a Strong Modifier of the Metabolic Impairments in Peritoneal Macrophages of Abcd1-Deficient Mice

The inherited peroxisomal disorder X-linked adrenoleukodystrophy (X-ALD), associated with neurodegeneration and inflammatory cerebral demyelination, is caused by mutations in the ABCD1 gene encoding the peroxisomal ATP-binding cassette (ABC) transporter ABCD1 (ALDP). ABCD1 transports CoA-esters of very long-chain fatty acids (VLCFA) into peroxisomes for degradation by β-oxidation; thus, ABCD1 deficiency results in VLCFA accumulation. The closest homologue, ABCD2 (ALDRP), when overexpressed, compensates for ABCD1 deficiency in X-ALD fibroblasts and in Abcd1-deficient mice. Microglia/macrophages have emerged as important players in the progression of neuroinflammation. Human monocytes, lacking significant expression of ABCD2, display severely impaired VLCFA metabolism in X-ALD. Here, we used thioglycollate-elicited primary mouse peritoneal macrophages (MPMΦ) from Abcd1 and Abcd2 single- and double-deficient mice to establish how these mutations affect VLCFA metabolism. By quantitative RT-PCR, Abcd2 mRNA was about half as abundant as Abcd1 mRNA in wild-type and similarly abundant in Abcd1-deficient MPMΦ. VLCFA (C26∶0) accumulated about twofold in Abcd1-deficient MPMΦ compared with wild-type controls, as measured by gas chromatography-mass spectrometry. In Abcd2-deficient macrophages VLCFA levels were normal. However, upon Abcd1/Abcd2 double-deficiency, VLCFA accumulation was markedly increased (sixfold) compared with Abcd1-deficient MPMΦ. Elovl1 mRNA, encoding the rate-limiting enzyme for elongation of VLCFA, was equally abundant across all genotypes. Peroxisomal β-oxidation of C26∶0 amounted to 62% of wild-type activity in Abcd1-deficient MPMΦ and was significantly more impaired (29% residual activity) upon Abcd1/Abcd2 double-deficiency. Single Abcd2 deficiency did not significantly compromise β-oxidation of C26∶0. Thus, the striking accumulation of VLCFA in double-deficient MPMΦ compared with single Abcd1 deficiency was due to the loss of ABCD2-mediated, compensatory transport of VLCFA into peroxisomes. We propose that moderate endogenous expression of Abcd2 in Abcd1-deficient murine macrophages prevents the severe metabolic phenotype observed in human X-ALD monocytes, which lack appreciable expression of ABCD2. This supports upregulation of ABCD2 as a therapeutic concept in X-ALD.     

Effect of dietary polyunsaturated fatty acids on the expression of peroxisomal ABC transporters

Peroxisomal ABC transporters encoded by the ABCD genes are thought to participate in the import of specific fatty acids in the peroxisomal matrix. ABCD1 deficiency is associated with X-linked adrenoleukodystrophy (X-ALD), the most frequent peroxisomal disorder which is characterized by the accumulation of saturated very-long-chain fatty acids (VLCFA). ABCD2 (the closest homolog of ABCD1) and ABCD3 have been shown to have partial functional redundancy with ABCD1; only when overexpressed, they can compensate for VLCFA accumulation. Other lipids, for instance polyunsaturated fatty acids (PUFA), should be possible candidate substrates for the ABCD2 and ABCD3 gene products, ALDRP and PMP70 respectively. Moreover, PUFA, which are known regulators of gene expression, could therefore represent potent inducers of the ABCD genes. To test this hypothesis, littermates of n-3-deficient rats were subjected to an n-3-deficient diet or equilibrated diets containing ALA (alpha-linolenic acid, 18:3n-3) as unique source of n-3 fatty acids or ALA plus DHA (docosahexaenoic acid, 22:6n-3) at two different doses. We analyzed the expression of peroxisomal ABC transporters and of the peroxisomal acyl-CoA oxidase gene 1 (Acox1) in adrenals, brain and liver. Whatever the diet, we did not observe any difference in gene expression in adrenals and brain. However, the hepatic expression level of Abcd2 and Abcd3 genes was found to be significantly higher in the n-3-deficient rats than in the rats fed the ALA diet or the DHA supplemented diets. This was accompanied by important changes in hepatic fatty acid composition. In summary, the hepatic expression of Abcd2 and Abcd3 but not of Abcd1 and Abcd4 appears to be highly sensitive towards dietary PUFA. This difference could be linked to the substrate specificity of the peroxisomal ABC transporters and a specific involvement of Abcd2 and Abcd3 in PUFA metabolism.     

SIRT5 inhibits peroxisomal ACOX1 to prevent oxidative damage and is downregulated in liver cancer

Peroxisomes account for ~35% of total H₂O₂ generation in mammalian tissues. Peroxisomal ACOX1 (acyl‐CoA oxidase 1) is the first and rate‐limiting enzyme in fatty acid β‐oxidation and a major producer of H₂O₂. ACOX1 dysfunction is linked to peroxisomal disorders and hepatocarcinogenesis. Here, we show that the deacetylase sirtuin 5 (SIRT5) is present in peroxisomes and that ACOX1 is a physiological substrate of SIRT5. Mechanistically, SIRT5‐mediated desuccinylation inhibits ACOX1 activity by suppressing its active dimer formation in both cultured cells and mouse livers. Deletion of SIRT5 increases H₂O₂ production and oxidative DNA damage, which can be alleviated by ACOX1 knockdown. We show that SIRT5 downregulation is associated with increased succinylation and activity of ACOX1 and oxidative DNA damage response in hepatocellular carcinoma (HCC). Our study reveals a novel role of SIRT5 in inhibiting peroxisome‐induced oxidative stress, in liver protection, and in suppressing HCC development.     

Uridine Prevents Fenofibrate-Induced Fatty Liver

Uridine, a pyrimidine nucleoside, can modulate liver lipid metabolism although its specific acting targets have not been identified. Using mice with fenofibrate-induced fatty liver as a model system, the effects of uridine on liver lipid metabolism are examined. At a daily dosage of 400 mg/kg, fenofibrate treatment causes reduction of liver NAD⁺/NADH ratio, induces hyper-acetylation of peroxisomal bifunctional enzyme (ECHD) and acyl-CoA oxidase 1 (ACOX1), and induces excessive accumulation of long chain fatty acids (LCFA) and very long chain fatty acids (VLCFA). Uridine co-administration at a daily dosage of 400 mg/kg raises NAD⁺/NADH ratio, inhibits fenofibrate-induced hyper-acetylation of ECHD, ACOX1, and reduces accumulation of LCFA and VLCFA. Our data indicates a therapeutic potential for uridine co-administration to prevent fenofibrate-induced fatty liver.     

Histone Deacetylase Inhibitor Upregulates Peroxisomal Fatty Acid Oxidation and Inhibits Apoptotic Cell Death in Abcd1-Deficient Glial Cells

In X-ALD, mutation/deletion of ALD gene (ABCD1) and the resultant very long chain fatty acid (VLCFA) derangement has dramatically opposing effects in astrocytes and oligodendrocytes. While loss of Abcd1 in astrocytes produces a robust inflammatory response, the oligodendrocytes undergo cell death leading to demyelination in X-linked adrenoleukodystrophy (X-ALD). The mechanisms of these distinct pathways in the two cell types are not well understood. Here, we investigated the effects of Abcd1-knockdown and the subsequent alteration in VLCFA metabolism in human U87 astrocytes and rat B12 oligodendrocytes. Loss of Abcd1 inhibited peroxisomal β-oxidation activity and increased expression of VLCFA synthesizing enzymes, elongase of very long chain fatty acids (ELOVLs) (1 and 3) in both cell types. However, higher induction of ELOVL''s in Abcd1-deficient B12 oligodendrocytes than astrocytes suggests that ELOVL pathway may play a prominent role in oligodendrocytes in X-ALD. While astrocytes are able to maintain the cellular homeostasis of anti-apoptotic proteins, Abcd1-deletion in B12 oligodendrocytes downregulated the anti-apototic (Bcl-2 and Bcl-xL) and cell survival (phospho-Erk1/2) proteins, and upregulated the pro-apoptotic proteins (Bad, Bim, Bax and Bid) leading to cell loss. These observations provide insights into different cellular signaling mechanisms in response to Abcd1-deletion in two different cell types of CNS. The apoptotic responses were accompanied by activation of caspase-3 and caspase-9 suggesting the involvement of mitochondrial-caspase-9-dependent mechanism in Abcd1-deficient oligodendrocytes. Treatment with histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) corrected the VLCFA derangement both in vitro and in vivo, and inhibited the oligodendrocytes loss. These observations provide a proof-of principle that HDAC inhibitor SAHA may have a therapeutic potential for X-ALD.     

PARK6 PINK1 mutants are defective in maintaining mitochondrial membrane potential and inhibiting ROS formation of substantia nigra dopaminergic neurons

Mutations in PTEN-induced kinase 1 (PINK1) gene cause recessive familial type 6 of Parkinson's disease (PARK6). PINK1 is believed to exert neuroprotective effect on SN dopaminergic cells by acting as a mitochondrial Ser/Thr protein kinase. Autosomal recessive inheritance indicates the involvement of loss of PINK1 function in PARK6 pathogenesis. In the present study, confocal imaging of cultured SN dopaminergic neurons prepared from PINK1 knockout mice was performed to investigate physiological importance of PINK1 in maintaining mitochondrial membrane potential (ΔΨ_m) and mitochondrial morphology and test the hypothesis that PARK6 mutations cause the loss of PINK1 function. PINK1-deficient SN dopaminergic neurons exhibited a depolarized ΔΨ_m. In contrast to long thread-like mitochondria of wild-type neurons, fragmented mitochondria were observed from PINK1-null SN dopaminergic cells. Basal level of mitochondrial superoxide and oxidative stressor H₂O₂-induced ROS generation were significantly increased in PINK1-deficient dopaminergic neurons. Overexpression of wild-type PINK1 restored hyperpolarized ΔΨ_m and thread-like mitochondrial morphology and inhibited ROS formation in PINK1-null dopaminergic cells. PARK6 mutant (G309D), (E417G) or (CΔ145) PINK1 failed to rescue mitochondrial dysfunction and inhibit oxidative stress in PINK1-deficient dopaminergic neurons. Mitochondrial toxin rotenone-induced cell death of dopaminergic neurons was augmented in PINK1-null SN neuronal culture. These results indicate that PINK1 is required for maintaining normal ΔΨ_m and mitochondrial morphology of cultured SN dopaminergic neurons and exerts its neuroprotective effect by inhibiting ROS formation. Our study also provides the evidence that PARK6 mutant (G309D), (E417G) or (CΔ145) PINK1 is defective in regulating mitochondrial functions and attenuating ROS production of SN dopaminergic cells.     

Cytokine-Induced Accumulation of Very Long-Chain Fatty Acids in Rat C6 Glial Cells: Implication for X-Adrenoleukodystrophy

Abstract: X-Adrenoleukodystrophy (X-ALD) is an inherited metabolic disorder of very long-chain fatty acids (VLCFA) with subsequent manifestation of neuroinflammatory disease. To investigate the possible role of proinflammatory cytokines in the X-ALD disease process, we examined the effect of cytokines on the metabolism of VLCFA in C6 glial cells expressing oligodendrocyte-like properties. C6 glial cells under serum-free conditions were treated with different combinations of cytokines (tumor necrosis factor-α, interleukin-1β, interferon-γ) or cytokine with bacterial lipopolysaccharide (LPS). Cytokine-treated C6 cells had higher concentrations of VLCFA, measured as percent weight and also as C_26:0/C_22:0 ratio, which were 300–400% as compared with the controls. We also found increased levels of C_26:1 in cytokine-treated cells. The accumulation of VLCFA paralleled the decrease (35–55%) in peroxisomal β-oxidation activity and a 12- to 14-fold increase in the production of nitric oxide (NO). Individual cytokines were unable either to produce NO or to increase the levels of VLCFA in C6 cells. Inhibition of cytokine-induced NO production by l -N-methylarginine, an inhibitor of NO synthase (NOS), and N-acetylcysteine, an inhibitor of cytokine-mediated induction of inducible NOS, normalized the peroxisomal β-oxidation activity and the levels of VLCFA, suggesting a role for the proinflammatory cytokines and NO toxicity in the neuropathological changes associated with abnormal VLCFA metabolism (e.g., X-ALD). X-ALD is a peroxisomal disease having deficient oxidation of VLCFA, resulting in the excessive accumulation of VLCFA in all tissues but especially in brain. We observed greater increase in levels of VLCFA in the inflammatory region of ALD brain (in the demyelinating plaque and the area around the plaque) than in the normal-looking area away from the plaque; this also indicates that cytokines in the proinflammatory region may augment the VLCFA defect caused by the inherited abnormality in X-ALD brain. Although C6 glial cultured cells do not reflect the X-ALD model precisely, the observed relationship between the cytokine-induced inhibition of the oxidation of VLCFA, excessive accumulation of VLCFA, and excessive production of NO and their normalization by inhibitors of NOS in C6 glial cells suggests that NO-mediated toxicity may play a role in VLCFA-associated neuroinflammatory diseases (e.g., X-ALD).     

Targeted Deletion of the PEX2 Peroxisome Assembly Gene in Mice Provides a Model for Zellweger Syndrome, a Human Neuronal Migration Disorder

Zellweger syndrome is a peroxisomal biogenesis disorder that results in abnormal neuronal migration in the central nervous system and severe neurologic dysfunction. The pathogenesis of the multiple severe anomalies associated with the disorders of peroxisome biogenesis remains unknown. To study the relationship between lack of peroxisomal function and organ dysfunction, the PEX2 peroxisome assembly gene (formerly peroxisome assembly factor-1) was disrupted by gene targeting.

Homozygous PEX2-deficient mice survive in utero but die several hours after birth. The mutant animals do not feed and are hypoactive and markedly hypotonic. The PEX2-deficient mice lack normal peroxisomes but do assemble empty peroxisome membrane ghosts. They display abnormal peroxisomal biochemical parameters, including accumulations of very long chain fatty acids in plasma and deficient erythrocyte plasmalogens. Abnormal lipid storage is evident in the adrenal cortex, with characteristic lamellar–lipid inclusions. In the central nervous system of newborn mutant mice there is disordered lamination in the cerebral cortex and an increased cell density in the underlying white matter, indicating an abnormality of neuronal migration. These findings demonstrate that mice with a PEX2 gene deletion have a peroxisomal disorder and provide an important model to study the role of peroxisomal function in the pathogenesis of this human disease.

     

Very long chain fatty acid beta-oxidation by rat liver mitochondria and peroxisomes

Crude mitochondrial fractions were isolated by differential centrifugation of rat liver homogenates. Subfractionation of these fractions on self-generating continuous Percoll gradients resulted in clearcut separation of peroxisomes from mitochondria. Hexacosanoic acid beta-oxidation was present mainly in peroxisomal fractions whereas hexacosanoyl CoA oxidation was present in the mitochondrial as well as in the peroxisomal fractions. The presence of much greater hexacosanoyl CoA synthetase activity in the purified preparations of microsomes and peroxisomes compared to mitochondria, suggests that the synthesis of coenzyme A derivatives of very long chain fatty acids (VLCFA) is limited in mitochondria. We postulate that a specific VLCFA CoA synthetase may be required to effectively convert VLCFA to VLCFA CoA in the cell. This specific synthetase activity is absent from the mitochondrial membrane, but present in the peroxisomal and the microsomal membranes. We postulate that substrate specificity and the subcellular localization of the specific VLCFA CoA synthetase directs and regulates VLCFA oxidation in the cell.     

Role of ALDP (ABCD1) and mitochondria in X-linked adrenoleukodystrophy

Peroxisomal disorders have been associated with malfunction of peroxisomal metabolic pathways, but the pathogenesis of these disorders is largely unknown. X-linked adrenoleukodystrophy (X-ALD) is associated with elevated levels of very-long-chain fatty acids (VLCFA; C(>22:0)) that have been attributed to reduced peroxisomal VLCFA beta-oxidation activity. Previously, our laboratory and others have reported elevated VLCFA levels and reduced peroxisomal VLCFA beta-oxidation in human and mouse X-ALD fibroblasts. In this study, we found normal levels of peroxisomal VLCFA beta-oxidation in tissues from ALD mice with elevated VLCFA levels. Treatment of ALD mice with pharmacological agents resulted in decreased VLCFA levels without a change in VLCFA beta-oxidation activity. These data indicate that ALDP does not determine the rate of VLCFA beta-oxidation and that VLCFA levels are not determined by the rate of VLCFA beta-oxidation. The rate of peroxisomal VLCFA beta-oxidation in human and mouse fibroblasts in vitro is affected by the rate of mitochondrial long-chain fatty acid beta-oxidation. We hypothesize that ALDP facilitates the interaction between peroxisomes and mitochondria, resulting, when ALDP is deficient in X-ALD, in increased VLCFA accumulation despite normal peroxisomal VLCFA beta-oxidation in ALD mouse tissues. In support of this hypothesis, mitochondrial structural abnormalities were observed in adrenal cortical cells of ALD mice.     

Structural insight into the substrate specificity of acyl-CoA oxidase1 from Yarrowia lipolytica for short-chain dicarboxylyl-CoAs

Acyl-CoA oxidase (ACOX) plays an important role in fatty acid degradation. The enzyme catalyzes the first reaction in peroxisomal fatty acid β-oxidation by reducing acyl-CoA to 2-trans-enoyl-CoA. The yeast Yarrowia lipolytica is able to utilize fatty acids, fats, and oil as carbon sources to produce valuable bioproducts. We determined the crystal structure of ACOX1 from Y. lipolytica (YlACOX1) at a resolution of 2.5 Å. YlACOX1 forms a homodimer, and the monomeric structure is composed of four domains, the Nα, Nβ, Cα1, and Cα2. The FAD cofactor is bound at the dimerization interface between the Nβ- and Cα1-domains. The substrate-binding tunnel formed by the interface between the Nα-, Nβ-, and Cα1-domains is located proximal to FAD. Amino acid and structural comparisons of YlACOX1 with other ACOXs show that the substrate-binding pocket of YlACOX1 is much smaller than that of the medium- or long-chain ACOXs but is rather similar to that of the short-chain ACOXs. Moreover, the hydrophilicity of residues constituting the end region of the substrate-binding pocket in YlACOX1 is quite similar to those in the short-chain ACOXs but different from those of the medium- or long-chain ACOXs. These observations provide structural insights how YlACOX1 prefers short-chain dicarboxylyl-CoAs as a substrate.     

Very-long-chain polyunsaturated fatty acids accumulate in phosphatidylcholine of fibroblasts from patients with Zellweger syndrome and acyl-CoA oxidase1 deficiency

Peroxisomes are subcellular organelles that function in multiple anabolic and catabolic processes, including β-oxidation of very-long-chain fatty acids (VLCFA) and biosynthesis of ether phospholipids. Peroxisomal disorders caused by defects in peroxisome biogenesis or peroxisomal β-oxidation manifest as severe neural disorders of the central nervous system. Abnormal peroxisomal metabolism is thought to be responsible for the clinical symptoms of these diseases, but their molecular pathogenesis remains to be elucidated. We performed lipidomic analysis to identify aberrant metabolites in fibroblasts from patients with Zellweger syndrome (ZS), acyl-CoA oxidase1 (AOx) deficiency, D-bifunctional protein (D-BP) and X-linked adrenoleukodystrophy (X-ALD), as well as in peroxisome-deficient Chinese hamster ovary cell mutants. In cells deficient in peroxisomal biogenesis, plasmenylethanolamine was remarkably reduced and phosphatidylethanolamine was increased. Marked accumulation of very-long-chain saturated fatty acid and monounsaturated fatty acids in phosphatidylcholine was observed in all mutant cells. Very-long-chain polyunsaturated fatty acid (VLC-PUFA) levels were significantly elevated, whilst phospholipids containing docosahexaenoic acid (DHA, C22:6n-3) were reduced in fibroblasts from patients with ZS, AOx deficiency, and D-BP deficiency, but not in fibroblasts from an X-ALD patient. Because patients with AOx deficiency suffer from more severe symptoms than those with X-ALD, accumulation of VLC-PUFA and/or reduction of DHA may be associated with the severity of peroxisomal diseases.     

The peroxisomal protein import machinery displays a preference for monomeric substrates

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and transported by the shuttling receptor PEX5 to the peroxisomal membrane docking/translocation machinery, where they are translocated into the organelle matrix. Under certain experimental conditions this protein import machinery has the remarkable capacity to accept already oligomerized proteins, a property that has heavily influenced current models on the mechanism of peroxisomal protein import. However, whether or not oligomeric proteins are really the best and most frequent clients of this machinery remain unclear. In this work, we present three lines of evidence suggesting that the peroxisomal import machinery displays a preference for monomeric proteins. First, in agreement with previous findings on catalase, we show that PEX5 binds newly synthesized (monomeric) acyl-CoA oxidase 1 (ACOX1) and urate oxidase (UOX), potently inhibiting their oligomerization. Second, in vitro import experiments suggest that monomeric ACOX1 and UOX are better peroxisomal import substrates than the corresponding oligomeric forms. Finally, we provide data strongly suggesting that although ACOX1 lacking a peroxisomal targeting signal can be imported into peroxisomes when co-expressed with ACOX1 containing its targeting signal, this import pathway is inefficient.     

Caffeic acid phenethyl ester induces adrenoleukodystrophy (Abcd2) gene in human X-ALD fibroblasts and inhibits the proinflammatory response in Abcd1/2 silenced mouse primary astrocytes

X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by mutations in the ABCD1 gene. Accumulation of very long chain fatty acids (VLCFA) that have been attributed to reduced peroxisomal VLCFA β-oxidation activity are the hallmark of the disease. Overexpression of ABCD2 gene, the closest homolog of ABCD1, has been shown to compensate for ABCD1, thus correcting the VLCFA derangement. The accumulation of VLCFA leads to a neuroinflammatory disease process associated with demyelination of the cerebral white matter. The present study underlines the importance of caffeic acid phenethyl ester (CAPE) in inducing the expression of ABCD2 (ALDRP), and normalizing the peroxisomal β-oxidation as well as the levels of saturated and monounsaturated VLCFAs in cultured human skin fibroblasts of X-ALD patients. The expression of ELOVL1, the single elongase catalyzing the synthesis of both saturated VLCFA (C26:0) and mono-unsaturated VLCFA (C26:1), was also reduced by CAPE treatment. Importantly, CAPE upregulated Abcd2 expression and peroxisomal β-oxidation and lowered the VLCFA levels in Abcd1-deficient U87 astrocytes and B12 oligodendrocytes. In addition, using Abcd1/Abcd2-silenced mouse primary astrocytes we examined the effects of CAPE in VLCFA-induced inflammatory response. CAPE treatment decreased the inflammatory response as the expression of inducible nitric oxide synthase, inflammatory cytokine, and activation of NF-κB in Abcd1/Abcd2-silenced mouse primary astrocytes was reduced. The observations indicate that CAPE corrects both the metabolic disease of VLCFA as well as secondary inflammatory disease; therefore, it may be a potential drug candidate to be tested for X-ALD therapy in humans.     

A novel case of ACOX2 deficiency leads to recognition of a third human peroxisomal acyl-CoA oxidase

Peroxisomal acyl-CoA oxidases catalyze the first step of beta-oxidation of a variety of substrates broken down in the peroxisome. These include the CoA-esters of very long-chain fatty acids, branched-chain fatty acids and the C27-bile acid intermediates. In rat, three peroxisomal acyl-CoA oxidases with different substrate specificities are known, whereas in humans it is believed that only two peroxisomal acyl-CoA oxidases are expressed under normal circumstances. Only three patients with ACOX2 deficiency, including two siblings, have been identified so far, showing accumulation of the C27-bile acid intermediates. Here, we performed biochemical studies in material from a novel ACOX2-deficient patient with increased levels of C27-bile acids in plasma, a complete loss of ACOX2 protein expression on immunoblot, but normal pristanic acid oxidation activity in fibroblasts. Since pristanoyl-CoA is presumed to be handled by ACOX2 specifically, these findings prompted us to re-investigate the expression of the human peroxisomal acyl-CoA oxidases. We report for the first time expression of ACOX3 in normal human tissues at the mRNA and protein level. Substrate specificity studies were done for ACOX1, 2 and 3 which revealed that ACOX1 is responsible for the oxidation of straight-chain fatty acids with different chain lengths, ACOX2 is the only human acyl-CoA oxidase involved in bile acid biosynthesis, and both ACOX2 and ACOX3 are involved in the degradation of the branched-chain fatty acids. Our studies provide new insights both into ACOX2 deficiency and into the role of the different acyl-CoA oxidases in peroxisomal metabolism.     

Genetic Dissection of Peroxisome-Associated Matrix Protein Degradation in Arabidopsis thaliana

Peroxisomes are organelles that sequester certain metabolic pathways; many of these pathways generate H₂O₂, which can damage proteins. However, little is known about how damaged or obsolete peroxisomal proteins are degraded. We exploit developmentally timed peroxisomal content remodeling in Arabidopsis thaliana to elucidate peroxisome-associated protein degradation. Isocitrate lyase (ICL) is a peroxisomal glyoxylate cycle enzyme necessary for early seedling development. A few days after germination, photosynthesis begins and ICL is degraded. We previously found that ICL is stabilized when a peroxisome-associated ubiquitin-conjugating enzyme and its membrane anchor are both mutated, suggesting that matrix proteins might exit the peroxisome for ubiquitin-dependent cytosolic degradation. To identify additional components needed for peroxisome-associated matrix protein degradation, we mutagenized a line expressing GFP–ICL, which is degraded similarly to endogenous ICL, and identified persistent GFP-ICL fluorescence (pfl) mutants. We found three pfl mutants that were defective in PEROXIN14 (PEX14/At5g62810), which encodes a peroxisomal membrane protein that assists in importing proteins into the peroxisome matrix, indicating that proteins must enter the peroxisome for efficient degradation. One pfl mutant was missing the peroxisomal 3-ketoacyl-CoA thiolase encoded by the PEROXISOME DEFECTIVE1 (PED1/At2g33150) gene, suggesting that peroxisomal metabolism influences the rate of matrix protein degradation. Finally, one pfl mutant that displayed normal matrix protein import carried a novel lesion in PEROXIN6 (PEX6/At1g03000), which encodes a peroxisome-tethered ATPase that is involved in recycling matrix protein receptors back to the cytosol. The isolation of pex6-2 as a pfl mutant supports the hypothesis that matrix proteins can exit the peroxisome for cytosolic degradation.     

Stimulation of peroxisomal palmitoyl-CoA oxidase activity by ciprofibrate in hepatic cell lines: Comparative studies in Fao,MH1C1 and HepG2 cells

Summary— The response of two rat cell lines, Fao and MH₁C₁, and one human cell line, HepG2, to the peroxisome proliferator ciprofibrate, was studied. Using a fluorometric assay for palmitoyl-CoA oxidase, the dose- and time-dependent increase of this enzymatic activity was determined. From the lowest concentration (100 μM) stimulation is evident in the two rat cell lines. In the Fao line, the activity was stimulated reaching a seven-fold increase over the control level at 250 μM after 72 h of treatment. In the MH₁C₁ line, the maximum stimulation, four- to five-fold, was obtained at 250 and 500 μM after 72 h. In the HepG2 cell line, activity increased two-fold at 250 μM after 72 h reaching a three-fold increase at 1000 μM after 48 h. Ciprofibrate was more toxic to Fao cells than to MH₁C₁ and HepG2 cells which is also the order of the acyl-CoA oxidase stimulation by ciprofibrate. These preliminary results suggest that the two rat cell lines are appropriate for investigating the induction of peroxisomal β-oxidation enzymes and the expression of their genes. The HepG2 cell line is a complementary model for the study of interspecies differences in the response to peroxisomal proliferators and of the peroxisomal functions implied in the lipid metabolism of human liver.