ABSTRACT
INTRODUCTION: The identification of blood biomarkers appears to be a means of improving diagnosis accuracy in Parkinson's disease (PD) and atypical parkinsonian syndromes (APS). We, therefore, evaluate the performance of neurodegeneration, oxidative stress and lipid metabolism plasma biomarkers to distinguish PD from APS. METHODS: This was a monocentric study with a cross-sectional design. Plasma levels and discriminating power of neurofilament light chain (NFL), malondialdehyde (MDA) and 24S-Hydroxycholesterol (24S-HC) were assessed in patients with clinical diagnoses of PD or APS. RESULTS: In total, 32 PD cases and 15 APS cases were included. Mean disease durations were 4.75 years in PD group and 4.2 years in APS group. Plasma levels of NFL, MDA and 24S-HC differed significantly between the APS and PD groups (P=0.003; P=0.009; P=0.032, respectively). NFL, MDA and 24S-HC discriminated between PD and APS (AUC=0.76688; AUC=0.7375; AUC=0.6958, respectively). APS diagnosis significantly increased with MDA level≥23.628nmol/mL (OR: 8.67, P=0.001), NFL level≥47.2pg/mL (OR: 11.92, P<0.001) or 24S-HC level≤33.4pmol/mL (OR: 6.17, P=0.008). APS diagnosis considerably increased with the combination of NFL and MDA levels beyond cutoff values (OR: 30.67, P<0.001). Finally, the combination of NFL and 24S-HC levels, or MDA and 24S-HC levels, or all three biomarkers' levels beyond cutoff values systematically classified patients in the APS group. CONCLUSION: Our results suggest that 24S-HC and especially MDA and NFL could be helpful for differentiating PD from APS. Further studies will be needed to reproduce our findings on larger, prospective cohorts of patients with parkinsonism evolving for less than 3 years.
Subject(s)
Parkinson Disease , Parkinsonian Disorders , Humans , Parkinson Disease/diagnosis , Parkinson Disease/metabolism , Prospective Studies , Cross-Sectional Studies , Lipid Metabolism , Parkinsonian Disorders/diagnosis , Biomarkers , Oxidative StressABSTRACT
Age-related diseases for which there are no effective treatments include cardiovascular diseases; neurodegenerative diseases such as Alzheimer's disease; eye disorders such as cataract and age-related macular degeneration; and, more recently, Severe Acute Respiratory Syndrome (SARS-CoV-2). These diseases are associated with plasma and/or tissue increases in cholesterol derivatives mainly formed by auto-oxidation: 7-ketocholesterol, also known as 7-oxo-cholesterol, and 7ß-hydroxycholesterol. The formation of these oxysterols can be considered as a consequence of mitochondrial and peroxisomal dysfunction, leading to increased in oxidative stress, which is accentuated with age. 7-ketocholesterol and 7ß-hydroxycholesterol cause a specific form of cytotoxic activity defined as oxiapoptophagy, including oxidative stress and induction of death by apoptosis associated with autophagic criteria. Oxiaptophagy is associated with organelle dysfunction and in particular with mitochondrial and peroxisomal alterations involved in the induction of cell death and in the rupture of redox balance. As the criteria characterizing 7-ketocholesterol- and 7ß-hydroxycholesterol-induced cytotoxicity are often simultaneously observed in major age-related diseases (cardiovascular diseases, age-related macular degeneration, Alzheimer's disease) the involvement of these oxysterols in the pathophysiology of the latter seems increasingly likely. It is therefore important to better understand the signalling pathways associated with the toxicity of 7-ketocholesterol and 7ß-hydroxycholesterol in order to identify pharmacological targets, nutrients and synthetic molecules attenuating or inhibiting the cytotoxic activities of these oxysterols. Numerous natural cytoprotective compounds have been identified: vitamins, fatty acids, polyphenols, terpenes, vegetal pigments, antioxidants, mixtures of compounds (oils, plant extracts) and bacterial enzymes. However, few synthetic molecules are able to prevent 7-ketocholesterol- and/or 7ß-hydroxycholesterol-induced cytotoxicity: dimethyl fumarate, monomethyl fumarate, the tyrosine kinase inhibitor AG126, memantine, simvastatine, Trolox, dimethylsufoxide, mangafodipir and mitochondrial permeability transition pore (MPTP) inhibitors. The effectiveness of these compounds, several of which are already in use in humans, makes it possible to consider using them for the treatment of certain age-related diseases associated with increased plasma and/or tissue levels of 7-ketocholesterol and/or 7ß-hydroxycholesterol.
Subject(s)
COVID-19 , Aging , Humans , Hydroxycholesterols , Ketocholesterols , Nutrients , Oils , SARS-CoV-2Subject(s)
Food Technology , Lipidomics , Oxysterols/pharmacology , Sterols/pharmacology , Biomedical Research/trends , Congresses as Topic , Cooperative Behavior , Food Technology/methods , Food Technology/trends , Humans , Internationality , Italy , Lipidomics/methods , Lipidomics/trends , Oxysterols/chemistry , Oxysterols/metabolism , Sterols/chemistry , Sterols/metabolismABSTRACT
Steroidal maleic anhydrides were prepared in one step: lithocholic, chenodeoxicholic, deoxicholic, ursocholic, and hyodeoxicholic acid derivatives. Their capability to induce cell death was studied on C6 rat glioma cells, and 7ß-hydroxycholesterol was used as positive cytotoxic control. The highest cytotoxicity was observed with lithocholic and chenodeoxicholic acid derivatives (23-(4-methylfuran-2,5-dione)-3α-hydroxy-24-nor-5ß-cholane (compound 1a), and 23-(4-methylfuran-2,5-dione)-3α,7α-dihydroxy-24-nor-5ß-cholane (compound 1b), respectively), which induce a non-apoptotic mode of cell death associated with mitochondrial membrane potential loss and reactive oxygen species overproduction. No cells with condensed and/or fragmented nuclei, no PARP degradation and no cleaved-caspase-3, which are apoptotic criteria, were observed. Similar effects were found with 7ß-hydroxycholesterol. The cell clonogenic survival assay showed that compound 1b was more cytotoxic than compound 1a and 7ß-hydroxycholesterol. Compound 1b and 7ß-hydroxycholesterol also induce cell cycle modifications. In addition, compounds 1a and 1b, and 7ß-hydroxycholesterol favour the formation of large acidic vacuoles revealed by staining with acridine orange and monodansylcadaverine evocating autophagic vacuoles; they also induce an increased ratio of [LC3-II / LC3-I], and modify the expression of mTOR, Beclin-1, Atg12, and Atg5-Atg12 which is are autophagic criteria. The ratio [LC3-II / LC3-I] is also strongly modified by bafilomycin acting on the autophagic flux. Rapamycin, an autophagic inducer, and 3-methyladenine, an autophagic inhibitor, reduce and increase 7ß-hydroxycholesterol-induced cell death, respectively, supporting that 7ß-hydroxycholesterol induces survival autophagy. Alpha-tocopherol also strongly attenuates 7ß-hydroxycholesterol-induced cell death. However, rapamycin, 3-methyladenine, and α-tocopherol have no effect on compounds 1a and 1b-induced cell death. It is concluded that these compounds trigger a non apoptotic mode of cell death, involving the mitochondria and associated with several characteristics of autophagy.
Subject(s)
Antineoplastic Agents/pharmacology , Autophagy/drug effects , Glioma/drug therapy , Hydroxycholesterols/pharmacology , Maleic Anhydrides/pharmacology , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , Glioma/metabolism , Hydroxycholesterols/chemistry , Maleic Anhydrides/chemistry , Membrane Potential, Mitochondrial/drug effects , RatsABSTRACT
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.
Subject(s)
ATP Binding Cassette Transporter, Subfamily D, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily D/genetics , Adrenoleukodystrophy/genetics , ATP Binding Cassette Transporter, Subfamily D/metabolism , ATP Binding Cassette Transporter, Subfamily D, Member 1/metabolism , Adrenoleukodystrophy/metabolism , Adrenoleukodystrophy/pathology , Animals , CRISPR-Cas Systems , Cell Line , Fatty Acids/metabolism , Female , Gene Deletion , Mice, Inbred C57BL , Microglia/metabolism , Microglia/pathologyABSTRACT
Acyl-CoA oxidase 1 (ACOX1) deficiency is a rare and severe peroxisomal leukodystrophy associated with a very long-chain fatty acid (VLCFA) ß-oxidation defect. This neurodegenerative disease lacks relevant cell models to further decipher the pathomechanisms in order to identify novel therapeutic targets. Since peroxisomal defects in microglia appear to be a key component of peroxisomal leukodystrophies, we targeted the Acox1 gene in the murine microglial BV-2 cell line. Using CRISPR/Cas9 gene editing, we generated an Acox1-deficient cell line and validated the allelic mutations, which lead to the absence of ACOX1 protein and enzymatic activity. The activity of catalase, the enzyme degrading H2O2, was increased, likely in response to the alteration of redox homeostasis. The mutant cell line grew more slowly than control cells without obvious morphological changes. However, ultrastructural analysis revealed an increased number of peroxisomes and mitochondria associated with size reduction of mitochondria. Changes in the distribution of lipid droplets containing neutral lipids have been observed in mutant cells; lipid analysis revealed the accumulation of saturated and monounsaturated VLCFA. Besides, expression levels of genes encoding interleukin-1 beta and 6 (IL-1ß and IL-6), as well as triggering receptor expressed on myeloid cells 2 (Trem2) were found modified in the mutant cells suggesting modification of microglial polarization and phagocytosis ability. In summary, this Acox1-deficient cell line presents the main biochemical characteristics of the human disease and will serve as a promising model to further investigate the consequences of a specific microglial peroxisomal ß-oxidation defect on oxidative stress, inflammation and cellular functions.
Subject(s)
Acyl-CoA Oxidase/deficiency , Microglia/cytology , Models, Biological , Mutation , Neurodegenerative Diseases/genetics , Acyl-CoA Oxidase/genetics , Animals , CRISPR-Cas Systems , Cell Line , Cell Proliferation , Fatty Acids/metabolism , Fatty Acids, Unsaturated/metabolism , Gene Editing , Hydrogen Peroxide/metabolism , Mice , Microglia/metabolism , Oxidative StressABSTRACT
Iron oxide nanoparticles have the capability to cross Blood Brain Barrier (BBB) and hence are widely investigated for biomedical operations in the central nervous system. Before being used for the biomedical purpose, it is necessary to investigate its biocompatibility, dosimetry and biological interaction. In the present study, in-house synthesized superparamagnetic iron oxide nanoparticles (SPIONs) were functionalized using the polymer, PolyEthylene Glycol (PEG) and a fluorophore (Rhodamine). The interaction of these nanoparticles with murine oligodendrocytes 158N was studied using different assays. The nanoparticles were taken up by the cells via endocytosis and there was a dose-dependent increase in the intracellular iron content as revealed by flow cytometry, transmission electron microscopy and confocal microscopy. Nanoparticles remained stable inside cells even after 24â¯h. Cell sorting capacity using a magnet depended on the number of particles interact per cell. SPIONs exhibited good biocompatibility as no toxicological responses, including morphological changes, loss of viability, oxidative stress or inflammatory response (IL-1ß, IL-6 secretion) were observed. Together, these data show that the in-house synthesized SPIONs have no side effects on 158N cells, and constitute interesting tools for biomedical applications across brain, including cellular imaging and targeting.
Subject(s)
Ferric Compounds/chemistry , Inflammation/pathology , Magnetite Nanoparticles/chemistry , Oligodendroglia/cytology , Oxidative Stress , Animals , Cell Death , Cell Survival , Cells, Cultured , Mice , Particle Size , Surface PropertiesABSTRACT
Titanate nanotubes (TiONts) are promising agents for biomedical applications. Microglial activation and associated oxidative burst are major challenges in drug delivery applications across the brain. Here, TiONts were designed for drug delivery systems by functionalizing them with (3-aminopropyl) triethoxysilane (APTES), their interactions and biocompatibility were studied in vitro using murine microglial BV-2 cells. TiONts-APTES exposure resulted in increased ROS production and transient mitochondrial hyperpolarization. However, there was no indication of microglial proliferation in BV-2 cells as suggested by cell cycle analysis and morphology evaluation. The endocytosis as well as passive diffusion mediated TiONts-APTES internalization were proved by transmission electron microscopy (TEM) with and without amiloride, an endocytosis inhibiting agent. In addition, the TiONts-APTES exhibited good biocompatibility on microglial BV-2 cells as revealed by the plasma membrane integrity, lysosmal membrane integrity, morphology and viability analysis.
Subject(s)
Biocompatible Materials/toxicity , Materials Testing , Microglia/drug effects , Nanotubes/toxicity , Titanium/toxicity , Cell Line , Cell Membrane/drug effects , Cell Proliferation/drug effects , Endocytosis/drug effects , Humans , Lysosomes/drug effects , Membrane Potential, Mitochondrial/drug effects , Reactive Oxygen Species , Respiratory Burst/drug effectsABSTRACT
Some factors related to diet, such as trans fatty acids (TFA), are known to be involved in the progression of atherosclerosis in humans. Thus, the aim of our study was (i) to evaluate the effects of three dietary free fatty acids (FFA) (elaidic (EA), oleic (OA) and palmitic acid (PA)) on U937 human monocytes, and (ii) to study the eventual benefits of bezafibrate (BZF), a pan-agonist for PPAR isoforms (α, γ and δ) in U937 cells treated with FFA. Morphologic and functional changes were investigated by microscopic and flow cytometric methods. Cellular lipid content, lipid droplets and FA composition were identified and studied. All analyses were also realized in association with or without BZF. Contrary to OA and PA, EA slightly induced both propidium iodide-positive cells and mitochondrial depolarization. In addition, in contrast to OA and PA, EA induced only a slight increase in superoxide anion production. However, EA and OA promoted cytoplasmic lipid droplets accumulation. Only EA and OA significantly increased CD36 expression. It is noteworthy that BZF had a more or less pronounced protective effect against EA-, OA- and PA-induced side effects: BZF attenuated the impaired cell viability and inflammatory response, decreased superoxide anion production and prevented the accumulation of neutral and polar lipids. The effects were less pronounced with OA and PA than with EA. Altogether, our data revealed a benefit of BZF on the side effects induced especially with EA. It may thus be of interest in preventing the early stages of atherosclerotic plaque formation.
Subject(s)
Bezafibrate/pharmacology , Cytoprotection/drug effects , Lipid Droplets/drug effects , Monocytes/drug effects , Oleic Acids/adverse effects , Plaque, Atherosclerotic/chemically induced , Plaque, Atherosclerotic/prevention & control , Case-Control Studies , Coronary Disease/blood , Coronary Disease/metabolism , Coronary Disease/pathology , Foam Cells/drug effects , Foam Cells/metabolism , Humans , Lipid Droplets/metabolism , Monocytes/metabolism , Oleic Acids/blood , Plaque, Atherosclerotic/metabolism , U937 CellsABSTRACT
Increased levels of C22:0, C24:0 and C26:0 were found in cortical lesions of patients with Alzheimer's disease (AD). So, it was of interest to precise the cytotoxic effects of these fatty acids, and to determine whether docosahexaenoic acid (DHA), described to prevent AD, can attenuate their eventual side effects. Human neuronal SK-N-BE cells were cultured in the absence or presence of C22:0, C24:0 or C26:0 (0.1-20 µM) without or with DHA (50-150 µM). C22:0, C24:0 and C26:0 induce an inhibition of cell growth, a loss of Δψm, an overproduction of reactive oxygen species (ROS), a decrease of reduced glutathione, and a lipid peroxidation. DHA attenuates C22:0, C24:0 and C26:0 induced-mitochondrial dysfunctions and/or cell growth inhibition measured with MTT whatever the concentrations considered, whereas it can either decrease or amplify (especially at 150 µM) ROS overproduction. C22:0, C24:0 and C26:0 have neurotoxic activities, and depending on its concentration, DHA attenuates or not fatty acid-induced side effects.
Subject(s)
Docosahexaenoic Acids/pharmacology , Fatty Acids/adverse effects , Mitochondria/drug effects , Mitochondria/pathology , Neurons/drug effects , Neurons/pathology , Oxidative Stress/drug effects , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Cell Line , Cell Proliferation/drug effects , Fatty Acids/metabolism , Glutathione/metabolism , Humans , Lipid Peroxidation/drug effects , Mitochondria/metabolism , Neurons/metabolism , Reactive Oxygen Species/metabolismABSTRACT
Cholesterol oxide derivatives (oxysterols) are viewed as potential biomarkers of neurodegenerative diseases. 24S-hydroxycholesterol, an oxysterol produced only in brain neurons, is often found for unknown reasons in increased levels in the plasma in patients with neurodegenerative diseases. On human neuronal SK-N-BE cells treated with hexacosanoic acid (C26:0) identified at increased levels in the tissues and plasma of patients with peroxisomal leukodystrophies and Alzheimer's disease, we observed increased level of 24S-hydroxycholesterol associated with C26:0 induced lipotoxicity. This finding reinforces the hypothesis suggesting that 24S-hydroxycholesterol could constitute a biomarker of neurotoxicity.
Subject(s)
Fatty Acids/pharmacology , Hydroxycholesterols/metabolism , Lipids/toxicity , Neurons/drug effects , Neurons/metabolism , Neurotoxicity Syndromes/diagnosis , Alzheimer Disease/etiology , Alzheimer Disease/metabolism , Animals , Biomarkers/metabolism , Brain/drug effects , Brain/metabolism , Cell Line , Cholesterol/metabolism , Humans , Neurotoxicity Syndromes/metabolismABSTRACT
In spite of the novel strategies to treat colon cancer, mortality rates associated with this disease remain consistently high. Tumour recurrence has been linked to the induction of resistance towards chemotherapy that involves cellular events that enable cancer cells to escape cell death. Treatment of colon cancer mainly implicates direct or indirect DNA-damaging agents and increased repair or tolerances towards subsequent lesions contribute to generate resistant populations. Resveratrol (RSV), a potent chemosensitising polyphenol, might share common properties with chemotherapeutic drugs through its indirect DNA-damaging effects reported in vitro. In this study, we investigated how RSV exerts its anticancer effects in models of colon cancer with a particular emphasis on the DNA-damage response (DDR; PIKKs-Chks-p53 signalling cascade) and its cellular consequences. We showed in vitro and in vivo that colon cancer models could progressively escape the repeated pharmacological treatments with RSV. We observed for the first time that this response was correlated with transient activation of the DDR, of apoptosis and senescence. In vitro, a single treatment with RSV induced a DDR correlated with S-phase delay and apoptosis, but prolonged treatments led to transient micronucleations and senescence phenotypes associated with polyploidisation. Ultimately, stable resistant populations towards RSV displaying higher degrees of ploidy and macronucleation as compared to parental cells emerged. We linked these transient effects and resistance emergence to the abilities of these cells to progressively escape RSV-induced DNA damage. Finally, we demonstrated that this DNA damage was triggered by an overproduction of reactive oxygen species (ROS) against which cancer cells could adapt under prolonged exposure to RSV. This study provides a pre-clinical analysis of the long-term effects of RSV and highlights ROS as main agents in RSV's indirect DNA-damaging properties and consequences in terms of anticancer response and potent resistance emergence.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Colon/drug effects , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Reactive Oxygen Species/metabolism , Stilbenes/pharmacology , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cell Line, Tumor , Checkpoint Kinase 1 , Checkpoint Kinase 2/genetics , Checkpoint Kinase 2/metabolism , Colon/metabolism , Colon/pathology , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , DNA Damage , Histones/genetics , Histones/metabolism , Humans , Polyploidy , Protein Kinases/genetics , Protein Kinases/metabolism , Rats , Resveratrol , S Phase/drug effects , S Phase/genetics , Signal Transduction , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Mussels may concentrate pollutants, with possibly significant side effects on human health. Therefore, mussels (Mytilus galloprovincialis) from two sites of the Moroccan Atlantic coast (Jorf Lasfar [JL], an industrial site, and Oualidia [OL], a vegetable-growing area), were subjected to biochemical analyses to quantify the presence of heavy metals (Cd, Cr, and Pb) and to establish the lipid profile: fatty acid, cholesterol, oxysterol, phytosterol and phospholipid content. In addition, mussel lipid extracts known to accumulate numerous toxic components were tested on murine pancreatic ß-cells (MIN6), and their biological activities were measured with various flow cytometric and biochemical methods to determine their impacts on cell death induction, organelle dysfunctions (mitochondria, lysosomes, and peroxisomes), oxidative stress and insulin secretion. The characteristics of JL and OL lipid extracts were compared with those of commercially available mussels from Spain (SP) used for human consumption. OL and JL contained heavy metals, high amounts of phospholipids, and high levels of oxysterols; the [(unsaturated fatty acids)/(saturated fatty acids)] ratio, which can be considered a sign of environmental stress leading to lipid peroxidation, was low. On MIN6 cells, JL and OL lipid extracts were able to trigger cell death. This event was associated with overproduction of H2 O2 , increased catalase activity, a decreased GSH level, lipid peroxidation and stimulation of insulin secretion. These effects were not observed with SP lipid extracts. These data suggest that some components from OL and JL lipid extracts might predispose to pancreatic dysfunctions. Epidemiological studies would be needed to assess the global risk on human health and the metabolic disease incidence in a context of regular seafood consumption from the OL and JL areas.
Subject(s)
Cadmium/toxicity , Chromium/toxicity , Insulin-Secreting Cells/drug effects , Lead/toxicity , Lipid Metabolism , Mytilus/metabolism , Animals , Cadmium/metabolism , Catalase/metabolism , Chromium/metabolism , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/metabolism , Lead/metabolism , Lipid Peroxidation/drug effects , Mice , Morocco , Oxidative Stress/drug effects , Spain , Tissue Extracts/metabolismABSTRACT
Peroxisomes are essential organelles in higher eukaryotes as they play a major role in numerous metabolic pathways and redox homeostasis. Some peroxisomal abnormalities, which are often not compatible with life or normal development, were identified in severe demyelinating and neurodegenerative brain diseases. The metabolic roles of peroxisomes, especially in the brain, are described and human brain peroxisomal disorders resulting from a peroxisome biogenesis or a single peroxisomal enzyme defect are listed. The brain abnormalities encountered in these disorders (demyelination, oxidative stress, inflammation, cell death, neuronal migration, differentiation) are described and their pathogenesis are discussed. Finally, the contribution of peroxisomal dysfunctions to the alterations of brain functions during aging and to the development of Alzheimer's disease is considered.
Subject(s)
Brain/metabolism , Peroxisomal Disorders/physiopathology , Peroxisomes/metabolism , Aging , Alzheimer Disease/physiopathology , Animals , Cell Death , Cell Differentiation/physiology , Cell Movement , Demyelinating Diseases/physiopathology , Encephalitis/physiopathology , Humans , Mice , Neurodegenerative Diseases/metabolism , Oxidative StressABSTRACT
X-linked adrenoleukodystrophy (X-ALD) and pseudo neonatal adrenoleukodystrophy (P-NALD) are neurodegenerative demyelinating diseases resulting from the functional loss of the peroxisomal ATP-binding cassette transporter D (ABCD1) and from single peroxisomal enzyme deficiency (Acyl-CoA oxidase1: ACOX1), respectively. As these proteins are involved in the catabolism of very long chain fatty acids (VLCFA: C24:0, C26:0), X-ALD and P-NALD patients are characterized by the accumulation of VLCFA in plasma and tissues. Since peroxisomes are involved in the metabolism of reactive oxygen species (ROS) and nitrogen species (RNS), we examined the impact of VLCFA on the oxidative status of 158N murine oligodendrocytes expressing or not Abcd1 or Acox1. VLCFA triggers an oxidative stress characterized by an overproduction of ROS and RNS associated with lipid peroxidation, protein carbonylation, increased superoxide dismutase (SOD) activity, decreased catalase activity and glutathione level. SiRNA knockdown of Abcd1 or Acox1 increased ROS and RNS production even in the absence of VLCFA, and especially potentialized VLCFA-induced ROS overproduction. Moreover, mainly in cells with reduced Acox1 level, the levels of VLCFA and neutral lipids were strongly enhanced both in untreated and VLCFA - treated cells. Our data obtained on 158N murine oligodendrocytes highlight that VLCFA induce an oxidative stress, and demonstrate that Abcd1 or Acox1 knockdown contributes to disrupt RedOx equilibrium supporting a link between oxidative stress and the deficiency of Abcd1 or Acox1 peroxisomal proteins.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Acyl-CoA Oxidase/metabolism , Fatty Acids/metabolism , Oligodendroglia/metabolism , Oxidative Stress/physiology , RNA Interference , ATP Binding Cassette Transporter, Subfamily D, Member 1 , ATP-Binding Cassette Transporters/genetics , Acyl-CoA Oxidase/genetics , Adrenoleukodystrophy/genetics , Adrenoleukodystrophy/metabolism , Animals , Blotting, Western , Cells, Cultured , Fatty Acids/pharmacology , Flow Cytometry , Gas Chromatography-Mass Spectrometry , Gene Knockdown Techniques , Mice , Oligodendroglia/drug effects , Oxidation-Reduction , Peroxisomes/metabolism , Reactive Oxygen Species/metabolism , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
Among several peroxisomal neurodegenerative disorders, the pseudoneonatal adrenoleukodystrophy (P-NALD) is characterized by the acyl-coenzyme A oxidase 1 (ACOX1) deficiency, which leads to the accumulation of very-long-chain fatty acids (VLCFA) and inflammatory demyelination. However, the components of this inflammatory process in P-NALD remain elusive. In this study, we used transcriptomic profiling and PCR array analyses to explore inflammatory gene expression in patient fibroblasts. Our results show the activation of IL-1 inflammatory pathway accompanied by the increased secretion of two IL-1 target genes, IL-6 and IL-8 cytokines. Human fibroblasts exposed to very-long-chain fatty acids exhibited increased mRNA expression of IL-1α and IL-1ß cytokines. Furthermore, expression of IL-6 and IL-8 cytokines in patient fibroblasts was down-regulated by MAPK, p38MAPK, and Jun N-terminal kinase inhibitors. Thus, the absence of acyl-coenzyme A oxidase 1 activity in P-NALD fibroblasts triggers an inflammatory process, in which the IL-1 pathway seems to be central. The use of specific kinase inhibitors may permit the modulation of the enhanced inflammatory status.
Subject(s)
Acyl-CoA Oxidase/genetics , Fibroblasts/metabolism , Inflammation/genetics , Transcriptome , Acyl-CoA Oxidase/deficiency , Acyl-CoA Oxidase/metabolism , Cells, Cultured , Fatty Acids/pharmacology , Fibroblasts/drug effects , Fibroblasts/pathology , Gene Expression Regulation/drug effects , Humans , Immunohistochemistry , Inflammation/metabolism , Inflammation Mediators/metabolism , Interleukin-1/genetics , Interleukin-1/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Interleukin-8/genetics , Interleukin-8/metabolism , Oligonucleotide Array Sequence Analysis , Osteopontin/genetics , Osteopontin/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
A pterygium is characterized by abnormal fibrovascular corneoconjunctival tissue. A number of investigations have attempted to elucidate this incompletely understood pathology. Since vascular endothelial growth factor (VEGF) and p53 are known to participate in tumor vascularization, our purpose was to study VEGF and p53 expression in active primary and recurrent pterygium from Tunisian patients. To this end, 15 cases of active primary pterygium and five cases of recurrent pterygium from Tunisia were studied by immunohistochemistry. Antibodies raised against VEGF and p53 were used to analyze the distribution and expression of these markers in pterygium and normal human conjunctiva were used as negative control. VEGF and p53 proteins were found in all cases of primary pterygium in epithelial, fibroblast and vascular endothelial cells. Active primary and recurrent pterygium have different patterns of expression. In primary pterygium, an important variability of p53 and VEGF expression was observed. However, in recurrent pterygium, p53 immunoreactivity was weak to moderate, whereas VEGF immunoreactivity was strong. In normal human conjunctiva, VEGF and p53 expression was weak to negative. The overexpression of VEGF in active primary and recurrent pterygium suggests that angiogenesis may play a role in pterygium pathogenesis and the expression of p53 in active primary pterygium, which might be associated with its mutated form, supports the hypothesis that actinic radiation may be involved in the genesis of pterygium. Thus, VEGF and p53 may be useful biomarkers for understanding the physiopathology of pterygium.
Subject(s)
Corneal Neovascularization/genetics , Pterygium/metabolism , Vascular Endothelial Growth Factor A/biosynthesis , Adult , Aged , Conjunctiva/metabolism , Female , Genes, p53 , Humans , Immunoenzyme Techniques , Male , Middle Aged , Pterygium/epidemiology , Pterygium/genetics , Recurrence , Tumor Suppressor Protein p53/biosynthesis , Tunisia/epidemiology , Vascular Endothelial Growth Factor A/genetics , Young AdultABSTRACT
Research of new diagnosis or prognosis biomarkers is a major challenge for the management of patients with complex pathologies like cancer. Clinical proteomics is one of the recent approaches to identify these biomarkers in biological fluids. Over the last five years, many problems related to the variability and the quality control of these analyses have been observed. This was notably related to the different preanalytical status of each sample. A strong need for standardization of the critical preanalytical phases (collection, transport, processing, storage...) has been therefore recognized. With this goal in mind, working groups of the "Institut national du cancer" (INCa) and the "Société française de biologie clinique" (SFBC) proposed here preanalytical proteomics guidelines for the most common biological fluids: plasma, serum, urine and cerebrospinal fluid. To goal is to provide the basis for the harmonization of the procedures in clinical laboratories and biobanks to allow an optimal use of biological collections.
Subject(s)
Body Fluids/physiology , Clinical Laboratory Techniques/standards , Diagnostic Techniques and Procedures/standards , Practice Guidelines as Topic , Proteomics/methods , Blood Chemical Analysis/standards , Humans , Prognosis , Proteinuria/diagnosis , Proteomics/standards , Urine/chemistryABSTRACT
The SFBC Working Group on << Preanalytics and multiplex analyses in proteomics >> is presenting a protocol which will allow harmonization of biospecimen research studies on the impact of different preanalytical variations on peptidic and protein analytes. This protocol is based upon standardization of preanalytical options corresponding to different preanalytical variations and different types of biospecimens (serum, plasma, cedrebrospinal fluid and urine). Application of this protocol will allow, not only harmonization of Biospecimen research, but also elaboration of standard nomenclature of the preanalytical steps.
Subject(s)
Peptides/analysis , Proteins/analysis , Proteomics/methods , Biological Specimen Banks/standards , Blood Chemical Analysis/standards , Cerebrospinal Fluid , Female , Humans , Male , Proteomics/standards , Specimen Handling/standards , Terminology as Topic , UrineABSTRACT
This manuscript reports the conclusions of a working group of the French Society of Clinical Biology (SFBC) 2007-2008 which evaluated the status, the impact and the future developments related to the analysis of the proteome using multiplexed methods. These approaches that are also used in clinical biology are performed on solid supports or in flow by using specifically dedicated or conventional flow cytometers. This review provides therefore a broad overview going from the methods already present in research laboratories to the tools for clinical and medical investigations.