Amira Zarrouk

Involvement of oxysterols in age-related diseases and ageing processes.

Ageing is accompanied by increasing vulnerability to major pathologies (atherosclerosis, Alzheimers disease, age-related macular degeneration, cataract, and osteoporosis) which can have similar underlying pathoetiologies. All of these diseases involve oxidative stress, inflammation and/or cell death processes, which are triggered by cholesterol oxide derivatives, also named oxysterols. These oxidized lipids result either from spontaneous and/or enzymatic oxidation of cholesterol on the steroid nucleus or on the side chain. The ability of oxysterols to induce severe dysfunctions in organelles (especially mitochondria) plays key roles in RedOx homeostasis, inflammatory status, lipid metabolism, and in the control of cell death induction, which may at least in part contribute to explain the potential participation of these molecules in ageing processes and in age related diseases. As no efficient treatments are currently available for most of these diseases, which are predicted to become more prevalent due to the increasing life expectancy and average age, a better knowledge of the biological activities of the different oxysterols is of interest, and constitutes an important step toward identification of pharmacological targets for the development of new therapeutic strategies.

Evidence of oxidative stress in very long chain fatty acid--treated oligodendrocytes and potentialization of ROS production using RNA interference-directed knockdown of ABCD1 and ACOX1 peroxisomal proteins.

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.

Induction of Mitochondrial Changes Associated with Oxidative Stress on Very Long Chain Fatty Acids (C22:0, C24:0, or C26:0)-Treated Human Neuronal Cells (SK-NB-E)

In Alzheimers disease, lipid alterations point towards peroxisomal dysfunctions. Indeed, a cortical accumulation of saturated very long chain fatty acids (VLCFAs: C22:0, C24:0, C26:0), substrates for peroxisomal β-oxidation, has been found in Alzheimer patients. This study was realized to investigate the effects of VLCFAs at the mitochondrial level since mitochondrial dysfunctions play crucial roles in neurodegeneration. On human neuronal SK-NB-E cells treated with C22:0, C24:0, or C26:0 (0.1–20 μM; 48 h), an inhibition of cell growth and mitochondrial dysfunctions were observed by cell counting with trypan blue, MTT assay, and measurement of mitochondrial transmembrane potential (Δψm) with DiOC6(3). A stimulation of oxidative stress was observed with DHE and MitoSOX used to quantify superoxide anion production on whole cells and at the mitochondrial level, respectively. With C24:0 and C26:0, by Western blotting, lower levels of mitochondrial complexes III and IV were detected. After staining with MitoTracker and by transmission electron microscopy used to study mitochondrial topography, mass and morphology, major changes were detected in VLCFAs treated-cells: modification of the cytoplasmic distribution of mitochondria, presence of large mitochondria, enhancement of the mitochondrial mass. Thus, VLCFAs can be potential risk factors contributing to neurodegeneration by inducing neuronal damages via mitochondrial dysfunctions.

Induction of oxiapoptophagy, a mixed mode of cell death associated with oxidative stress, apoptosis and autophagy, on 7-ketocholesterol-treated 158N murine oligodendrocytes: impairment by α-tocopherol.

7-Ketocholesterol (7KC) has been suggested to induce a complex mode of cell death on monocytic cells: oxiapoptophagy (OXIdation, APOPTOsis, and autoPHAGY) (Monier et al. (2003) [12]). The aim of the present study, realized on 158N murine oligodendrocytes, was to bring new evidence on this mixed form of cell death. On 158N cells, 7KC induces an overproduction of reactive oxygen species (ROS) revealed by dihydroethidium staining, a loss of transmembrane mitochondrial potential measured with DiOC6(3), caspase-3 activation, and condensation and/or fragmentation of the nuclei which are typical criteria of oxidative stress and apoptosis. Moreover, 7KC enhances cytoplamic membrane permeability to propidium iodide, and induces acidic vesicular organelle formation evaluated with acridine orange. In addition, 7KC promotes conversion of microtubule-associated protein light chain 3 (LC3-I) to LC3-II which is characteristic of autophagy. These different side effects were impaired by α-tocopherol. Altogether, our data demonstrate that oxiapoptophagy including ROS overproduction, apoptosis and autophagy could be a particular type of cell death activated by 7KC which can be inhibited by α-tocopherol.

Induction of oxiapoptophagy on 158N murine oligodendrocytes treated by 7-ketocholesterol-, 7β-hydroxycholesterol-, or 24(S)-hydroxycholesterol: Protective effects of α-tocopherol and docosahexaenoic acid (DHA; C22:6 n-3).

In demyelinating or non-demyelinating neurodegenerative diseases, increased levels of 7-ketocholesterol (7KC), 7β-hydroxycholesterol (7β-OHC) and 24(S)-hydroxycholesterol (24S-OHC) can be observed in brain lesions. In 158N murine oligodendrocytes, 7KC triggers a complex mode of cell death defined as oxiapoptophagy, involving simultaneous oxidative stress, apoptosis and autophagy. In these cells, 7KC as well as 7β-OHC and 24S-OHC induce a decrease of cell proliferation evaluated by phase contrast microscopy, an alteration of mitochondrial activity quantified with the MTT test, an overproduction of reactive oxygen species revealed by staining with dihydroethidium and dihydrorhodamine 123, caspase-3 activation, PARP degradation, reduced expression of Bcl-2, and condensation and/or fragmentation of the nuclei which are typical criteria of oxidative stress and apoptosis. Moreover, 7KC, 7β-OHC and 24S-OHC promote conversion of microtubule-associated protein light chain 3 (LC3-I) to LC3-II which is a characteristic of autophagy. Consequently, 7β-OHC and 24S-OHC, similarly to 7KC, can be considered as potent inducers of oxiapoptophagy. Furthermore, the different cytotoxic effects associated with 7KC, 7β-OHC and 24S-OHC-induced oxiapoptophagy are attenuated by vitamin E (VitE, α-tocopherol) and DHA which enhances VitE protective effects. In 158N murine oligodendrocytes, our data support the concept that oxiapoptophagy, which can be inhibited by VitE and DHA, could be a particular mode of cell death elicited by cytotoxic oxysterols.

Absence of correlation between oxysterol accumulation in lipid raft microdomains, calcium increase, and apoptosis induction on 158N murine oligodendrocytes

There is some evidence that oxidized derivatives of cholesterol, 7-ketocholesterol (7KC) and 7β-hydroxycholesterol (7βOHC), are increased in the plasma of patients with neurodegenerative diseases associated with demyelinization of the central nervous system (CNS). It was therefore of interest to investigate the effects of these oxysterols on oligodendrocytes, the myelin-forming cells in the CNS. To this end, 158N murine oligodendrocytes were treated with 7KC or 7βOHC inducing an apoptotic mode of cell death characterized by condensation/fragmentation of the nuclei, dephosphorylation of Akt and GSK3, mitochondrial depolarization involving Mcl-1, and caspase-3 activation. In contrast, under treatment with 27-hydroxycholesterol (27OHC), no cell death was observed. When the cells were stained with Fura-2, no significant Ca(2+) rise was found with the different oxysterols, whereas strong signals were detected with ionomycin used as positive control. At concentrations which induced apoptosis, 7KC but not 7βOHC accumulated in lipid rafts. Although not cytotoxic, 27OHC was mainly detected in lipid rafts. It is noteworthy that α-tocopherol (but not ellagic acid and resveratrol) was able to counteract 7KC- and 7βOHC-induced apoptosis and to decrease the accumulation of 7KC and 27OHC in lipid rafts. Thus, in 158N cells, the ability of oxysterols to trigger a mode of cell death by apoptosis involving GSK-3 and caspase-3 activation is independent of the increase in the Ca(2+) level and of their accumulation in lipid raft microdomains.

Effects of cholesterol oxides on cell death induction and calcium increase in human neuronal cells (SK-N-BE) and evaluation of the protective effects of docosahexaenoic acid (DHA; C22:6 n-3)

Some oxysterols are associated with neurodegenerative diseases. Their lipotoxicity is characterized by an oxidative stress and induction of apoptosis. To evaluate the capacity of these molecules to trigger cellular modifications involved in neurodegeneration, human neuronal cells SK-N-BE were treated with 7-ketocholesterol, 7α- and 7β-hydroxycholesterol, 6α- and 6β-hydroxycholesterol, 4α- and 4β-hydroxycholesterol, 24(S)-hydroxycholesterol and 27-hydroxycholesterol (50-100μM, 24h) without or with docosahexaenoic acid (50μM). The effects of these compounds on mitochondrial activity, cell growth, production of reactive oxygen species (ROS) and superoxide anions (O2(-)), catalase and superoxide dismutase activities were determined. The ability of the oxysterols to induce increases in Ca(2+) was measured after 10min and 24h of treatment using fura-2 videomicroscopy and Von Kossa staining, respectively. Cholesterol, 7-ketocholesterol, 7β-hydroxycholesterol, and 24(S)-hydroxycholesterol (100μM) induced mitochondrial dysfunction, cell growth inhibition, ROS overproduction and cell death. A slight increase in the percentage of cells with condensed and/or fragmented nuclei, characteristic of apoptotic cells, was detected. With 27-hydroxycholesterol, a marked increase of O2(-) was observed. Increases in intracellular Ca(2+) were only found with 7-ketocholesterol, 7β-hydroxycholesterol, 24(S)-hydroxycholesterol and 27-hydroxycholesterol. Pre-treatment with docosahexaenoic acid showed some protective effects depending on the oxysterol considered. According to the present data, 7-ketocholesterol, 7β-hydroxycholesterol, 24(S)-hydroxycholesterol and 27-hydroxycholesterol could favor neurodegeneration by their abilities to induce mitochondrial dysfunctions, oxidative stress and/or cell death associated or not with increases in cytosolic calcium levels.

Fatty Acid Profiles in Demented Patients: Identification of Hexacosanoic Acid (C26:0) as a Blood Lipid Biomarker of Dementia

BACKGROUND Several lipid metabolism alterations have been described in the brain and plasma of Alzheimers disease (AD) patients, suggesting a relation between lipid metabolism alteration and dementia. OBJECTIVE We attempted to identify blood fatty acids as biomarkers of dementia. METHODS Fatty acid profiles were established using gas chromatography with or without mass spectrometry on matched plasma and red blood cells (RBCs) of demented patients diagnosed with AD, vascular dementia, or other dementia, and compared with a control group of elderly individuals. The severity of dementia was evaluated with the Mini-Mental State Examination test. RESULTS Fatty acid analysis showed significant variations of fatty acid levels in demented patients including AD patients. The highest plasma and RBC accumulation was found with hexacosanoic acid (C26:0). Our data also support that alterations of desaturase and elongase activities may contribute to cognitive dysfunction. CONCLUSION The variations of fatty acid levels and the accumulation of C26:0 in the plasma and RBCs highlight an alteration of fatty acid metabolism in demented patients and point toward possible peroxisomal dysfunction. It is suggested that C26:0 may constitute a convenient blood biomarker of dementia that could be useful in routine medical practice.

7-Ketocholesterol is increased in the plasma of X-ALD patients and induces peroxisomal modifications in microglial cells: Potential roles of 7-ketocholesterol in the pathophysiology of X-ALD.

X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder induced by a mutation in the ABCD1 gene, which causes the accumulation of very long-chain fatty acids in tissue and plasma. Oxidative stress may be a hallmark of X-ALD. In the plasma of X-ALD patients with different forms of the disease, characterized by high levels of C24:0 and C26:0, we observed the presence of oxidative stress revealed by decreased levels of GSH, α-tocopherol, and docosahexaenoic acid (DHA). We showed that oxidative stress caused the oxidation of cholesterol and linoleic acid, leading to the formation of cholesterol oxide derivatives oxidized at C7 (7-ketocholesterol (7KC), 7β-hydroxycholesterol (7β-OHC), and 7α-hydroxycholesrol (7α-OHC)) and of 9- and 13-hydroxyoctadecadienoic acids (9-HODE, 13-HODE), respectively. High levels of 7KC, 7β-OHC, 7α-OHC, 9-HODE and 13-HODE were found. As 7KC induces oxidative stress, inflammation and cell death, which could play key roles in the development of X-ALD, the impact of 7KC on the peroxisomal status was determined in microglial BV-2 cells. Indeed, environmental stress factors such as 7KC could exacerbate peroxisomal dysfunctions in microglial cells and thus determine the progression of the disease. 7KC induces oxiapoptophagy in BV-2 cells: overproduction of H2O2 and O2-, presence of cleaved caspase-3 and PARP, nuclear condensation and/or fragmentation; elevated [LC3-II/LC3-I] ratio, increased p62 levels. 7KC also induces several peroxisomal modifications: decreased Abcd1, Abcd2, Abcd3, Acox1 and/or Mfp2 mRNA and protein levels, increased catalase activity and decreased Acox1-activity. However, the Pex14 level was unchanged. It is suggested that high levels of 7KC in X-ALD patients could foster generalized peroxisomal dysfunction in microglial cells, which could in turn intensify brain damage.

Protective Effects of α-Tocopherol, γ-Tocopherol and Oleic Acid, Three Compounds of Olive Oils, and No Effect of Trolox, on 7-Ketocholesterol-Induced Mitochondrial and Peroxisomal Dysfunction in Microglial BV-2 Cells

Lipid peroxidation products, such as 7-ketocholesterol (7KC), may be increased in the body fluids and tissues of patients with neurodegenerative diseases and trigger microglial dysfunction involved in neurodegeneration. It is therefore important to identify synthetic and natural molecules able to impair the toxic effects of 7KC. We determined the impact of 7KC on murine microglial BV-2 cells, especially its ability to trigger mitochondrial and peroxisomal dysfunction, and evaluated the protective effects of α- and γ-tocopherol, Trolox, and oleic acid (OA). Multiple complementary chemical assays, flow cytometric and biochemical methods were used to evaluate the antioxidant and cytoprotective properties of these molecules. According to various complementary assays to estimate antioxidant activity, only α-, and γ-tocopherol, and Trolox had antioxidant properties. However, only α-tocopherol, γ-tocopherol and OA were able to impair 7KC-induced loss of mitochondrial transmembrane potential, which is associated with increased permeability to propidium iodide, an indicator of cell death. In addition, α-and γ-tocopherol, and OA were able to prevent the decrease in Abcd3 protein levels, which allows the measurement of peroxisomal mass, and in mRNA levels of Abcd1 and Abcd2, which encode for two transporters involved in peroxisomal β-oxidation. Thus, 7KC-induced side effects are associated with mitochondrial and peroxisomal dysfunction which can be inversed by natural compounds, thus supporting the hypothesis that the composition of the diet can act on the function of organelles involved in neurodegenerative diseases.