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Biotin attenuation of oxidative stress, mitochondrial dysfunction, lipid metabolism alteration and 7β-hydroxycholesterol-induced cell death in 158N murine oligodendrocytes

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Abstract

Mitochondrial dysfunction and oxidative stress are involved in neurodegenerative diseases associated with an enhancement of lipid peroxidation products such as 7β- hydroxycholesterol (7β-OHC). It is therefore important to study the ability of 7β-OHC to trigger mitochondrial defects, oxidative stress, metabolic dysfunctions and cell death, which are hallmarks of neurodegeneration, and to identify cytoprotective molecules. The effects of biotin were evaluated on 158N murine oligodendrocytes, which are myelin synthesizing cells, exposed to 7β-OHC (50 µM) with or without biotin (10 and 100 nM) or α-tocopherol (positive control of cytoprotection). The effects of biotin on 7β-OHC activities were determined using different criteria: cell adhesion; plasma membrane integrity; redox status. The impact on mitochondria was characterized by the measurement of transmembrane mitochondrial potential (ΔΨm), reactive oxygen species (ROS) overproduction, mitochondrial mass, quantification of cardiolipins and organic acids. Sterols and fatty acids were also quantified. Cell death (apoptosis, autophagy) was characterized by the numeration of apoptotic cells, caspase-3 activation, identification of autophagic vesicles, and activation of LC3-I into LC3-II. Biotin attenuates 7β-OHC-induced cytotoxicity: loss of cell adhesion was reduced; antioxidant activities were normalized. ROS overproduction, protein and lipid oxidation products were decreased. Biotin partially restores mitochondrial functions: attenuation of the loss of ΔΨm; reduced levels of mitochondrial O2•− overproduction; normalization of cardiolipins and organic acid levels. Biotin also normalizes cholesterol and fatty acid synthesis, and prevents apoptosis and autophagy (oxiapoptophagy). Our data support that biotin, which prevents oligodendrocytes damages, could be useful in the treatment of neurodegeneration and demyelination.

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... Cui and colleagues reported that, in vitro, MD1003 did not increase the percentage of rat OPCs but increased the percentage of ensheathing O4+ oligodendrocytes in association with ATP production; yet, it failed to increase the percentage of MBP+ myelinating OLs three days following treatment [28]. Biotin also has the potential to attenuate murine OLs oxidative stress, mitochondria dysfunction and cell death [24]. Moreover, PLP and MBP expression were not, or only slightly affected under treatment with 10-100 ng biotin [24]. ...
... Biotin also has the potential to attenuate murine OLs oxidative stress, mitochondria dysfunction and cell death [24]. Moreover, PLP and MBP expression were not, or only slightly affected under treatment with 10-100 ng biotin [24]. These data together with our observations, suggest that MD1003 can enhance the differentiation potential of OPCs (likely providing metabolic support and favoring mitochondrial function) but not myelination. ...
... It is therefore possible that MD1003 alone was not sufficient to impact on myelination. When murine OLs (with mitochondrial defect and metabolic dysfunction) were exposed to biotin plus α-tocopherol (a known cytoprotective drug) in vitro, MBP expression was strongly enhanced (as compared to biotin or α-tocopherol treatment alone) [24] which suggests that combination therapies targeting can be beneficial to promote myelin repair. ...
Article
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Accumulating evidences suggest a strong correlation between metabolic changes and neurodegeneration in CNS demyelinating diseases such as multiple sclerosis (MS). Biotin, an essential cofactor for five carboxylases, is expressed by oligodendrocytes and involved in fatty acid synthesis and energy production. The metabolic effect of biotin or high-dose-biotin (MD1003) has been reported on rodent oligodendrocytes in vitro, and in neurodegenerative or demyelinating animal models. However, clinical studies, showed mild or no beneficial effect of MD1003 in amyotrophic lateral sclerosis (ALS) or MS. Here, we took advantage of a mouse model of myelin deficiency to study the effects of MD1003 on the behavior of murine and grafted human oligodendrocytes in vivo. We show that MD1003 increases the number and the differentiation potential of endogenous murine oligodendroglia over time. Moreover, the levels of MD1003 are increased in the plasma and brain of pups born to treated mothers, indicating that MD1003 can pass through the mother’s milk. The histological analysis of the grafted animals shows that MD1003 increased proliferation and accelerates differentiation of human oligodendroglia, but without enhancing their myelination potential. These findings provide important insights into the role of MD1003 on murine and human oligodendrocyte maturation/myelination that may explain the mitigated outcome of ALS/MS clinical trials.
... •− , and increase in the number and size of mitochondria, probably compensatory for their dysfunction [112,113]. ...
... In addition, peroxysomal dysfunction is responsible for further disequilibrium of the redox status, with inappropriate detoxification of ROS and dysregulation of protein and fatty acid oxidation [112]. Consequently, increased energy demand of demyelinated neurons cannot be compensated and results in their damage. ...
... The 4-HNE, present in elevated concentrations in foamy macrophages and astrocytes in active demyelinating MS lesions [150], along with ROS, were found to be detrimental to CNS cell viability, as well as BBB integrity and functionality [152]. Several studies have indicated that increased levels of ROS and lipid peroxidation products in the CSF and plasma of MS patients, along with mitochondrial damage, strongly reiterates the importance of oxidative damage in MS progression [112,147,150,153,154]. ...
Article
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Multiple sclerosis (MS) is a central nervous system disease with complex pathogenesis, including two main processes: immune-mediated inflammatory demyelination and progressive degeneration with axonal loss. Despite recent progress in our understanding and management of MS, availability of sensitive and specific biomarkers for these both processes, as well as neuroprotective therapeutic options targeted at progressive phase of disease, are still being sought. Given their abundance in the myelin sheath, lipids are believed to play a central role in underlying immunopathogenesis in MS and seem to be a promising subject of investigation in this field. On the basis of our previous research and a review of the literature, we discuss the current understanding of lipid-related mechanisms involved in active relapse, remission, and progression of MS. These insights highlight potential usefulness of lipid markers in prediction or monitoring the course of MS, particularly in its progressive stage, still insufficiently addressed. Furthermore, they raise hope for new, effective, and stage-specific treatment options, involving lipids as targets or carriers of therapeutic agents.
... Alongside lipids, proteins can also be damaged by ROS. The determination of carbonylated proteins (CP) level was determined spectrophotometrically as described previously [37]. Data are expressed as µmoles/mg of protein. ...
... Numerous studies reported pro-oxidative activities of oxysterols including 7β-OHC [37][38][39][40]. Thus, we intended to evaluate the ability of 7β-OHC (40 µg/mL) to induce intracellular ROS production in comparison with H2O2 (100 µM) using H2DCFDA fluorescence assay. ...
... These molecules can cross the blood-brain barrier and accumulate in the plasma membrane and inside the cell [42]. Some oxysterols, including 7β-OHC, exhibit pro-oxidative and pro-apoptotic properties [37][38][39][40]. ...
Article
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Oxysterols are assumed to be the driving force behind numerous neurodegenerative diseases. In this work, we aimed to study the ability of 7β-hydroxycholesterol (7β-OHC) to trigger oxidative stress and cell death in human neuroblastoma cells (SH-SY5Y) then the capacity of Nigella sativa and Milk thistle seed oils (NSO and MTSO respectively) to oppose 7β-OHC-induced side effects. The impact of 7β-OHC, associated or not with NSO or MTSO, was studied on different criteria: cell viability; redox status and apoptosis. Oxidative stress was assessed through the intracellular reactive oxygen species (ROS) production, levels of enzymatic and non-enzymatic antioxidants, lipidand protein oxidation products. Our results indicate that 7β-OHC [40 µg/mL] exhibit pr-oxidative and pro-apoptotic activities shown by a decrease of the antioxidant enzymatic activities and an increase of ROS production, lipid, and protein oxidation end products as well as nitrotyrosine formation and caspase 3 activation. However, under the pre-treatment with NSO, and especially with MTSO [100 µg/mL], a marked attenuation of oxidative damages was observed. Our study suggests harmful effects of 7β-OHC consisting of pro-oxidative,anti-proliferativeand pro-apoptotic activities that may contribute to neurodegeneration. NSO and especially MTSO showed potential cytoprotection against the cytotoxicity of 7β-OHC.
... catalase, SOD and GPx); enhanced levels of CDs, MDA and CPs; (Li et al., 2012;Zarrouk et al., 2015;Nury et al., 2020b). In addition, it induces dysfunction in several organelles, including morphological, topographical and functional changes of mitochondria, peroxisomes, and lysosomes (Sghaier et al., 2019a;Sghaier et al., 2019b;Nury et al., 2020a). The loss of ΔΨm resulting from mitochondrial dysfunction contributes to activation of apoptosis (Sghaier et al., 2019a;Sghaier et al., 2019b). ...
... In addition, it induces dysfunction in several organelles, including morphological, topographical and functional changes of mitochondria, peroxisomes, and lysosomes (Sghaier et al., 2019a;Sghaier et al., 2019b;Nury et al., 2020a). The loss of ΔΨm resulting from mitochondrial dysfunction contributes to activation of apoptosis (Sghaier et al., 2019a;Sghaier et al., 2019b). Moreover, 7β-OHC-induced cell death is associated with autophagic criteria: presence of large cytoplasmic vacuoles revealed by staining with monodansylcadaverine and activation of LC3-I into LC3-II (Sghaier et al., 2019a;Sghaier et al., 2019b). ...
... The loss of ΔΨm resulting from mitochondrial dysfunction contributes to activation of apoptosis (Sghaier et al., 2019a;Sghaier et al., 2019b). Moreover, 7β-OHC-induced cell death is associated with autophagic criteria: presence of large cytoplasmic vacuoles revealed by staining with monodansylcadaverine and activation of LC3-I into LC3-II (Sghaier et al., 2019a;Sghaier et al., 2019b). As 7β-OHC and 7KC are two potent inducers of oxiapoptophagy some signalling pathways are therefore similar. ...
Article
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 7β-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 7β-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 7β-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 7β-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 7β-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 7β-hydroxycholesterol.
... In 158 N cells, anti-apoptotic and anti-oxidant capacities of natural or synthetic compounds and their ability to attenuate or to prevent toxicity of 7KC or 7β-OHC were often evaluated. In 158 N cells incubated with 7KC or 7β-OHC, the cytoprotective effect of dimethylfumarate (DMF; Tecfidera) and biotin (vitamin B8), which are used for the treatment of MS, have been shown [120,121]. Natural products including argan oil, olive oil, milk thistle seed oil, sea urchin egg oil, Carpobrotus edulis ethanol-water extract and ethanolic mint extracts also strongly attenuate 7KC and/or 7β-OHCinduced cytotoxicity [87,[121][122][123][124][125]. ...
... In 158 N cells incubated with 7KC or 7β-OHC, the cytoprotective effect of dimethylfumarate (DMF; Tecfidera) and biotin (vitamin B8), which are used for the treatment of MS, have been shown [120,121]. Natural products including argan oil, olive oil, milk thistle seed oil, sea urchin egg oil, Carpobrotus edulis ethanol-water extract and ethanolic mint extracts also strongly attenuate 7KC and/or 7β-OHCinduced cytotoxicity [87,[121][122][123][124][125]. ...
... The schematic signaling pathways presented in Fig. 3 summarize the data obtained with 7β-OHC on cells from different types and species: promonocytic/monocytic human U937/THP1 cells [25,47]; vascular cells (endothelial and smooth muscle cells) [25,148]; wild type human mammary tumor MCF-7 cells (caspase-3 deficient), and genetically modified MCF-7 (MCF-7/c3: stably transfected with caspase-3) [138], human retinal pigment epithelial cells [100]/primary porcine retinal epithelial cells; rat C6 glioblastoma cells [170], murine oligodendrocytes 158 N and murine microglial BV-2 cells [3,114,119,121,141,142]. The different cells used permitted determination of the signaling pathways involved in 7β-OHC-induced cytotoxicity and to define the relationships between ROS overproduction, apoptosis, autophagy and inflammation. ...
Article
Oxysterols are molecules derived by the oxidation of cholesterol and can be formed either by auto-oxidation, enzymatically or by both processes. Among the oxysterols formed by auto-oxidation, 7-ketocholesterol and 7β-hydroxycholesterol are the main forms generated. These oxysterols, formed endogenously and brought in large quantities by certain foods, have major cytotoxic properties. They are powerful inducers of oxidative stress, inducing dysfunction of organelles (mitochondria, lysosomes and peroxisomes) that can cause cell death. These molecules are often identified in increased amounts in common pathological states such as cardiovascular diseases, certain eye conditions, neurodegenerative disorders and inflammatory bowel diseases. To oppose the cytotoxic effects of these molecules, it is important to know their biological activities and the signaling pathways they affect. Numerous cell models of the vascular wall, eye, brain, and digestive tract have been used. Currently, to counter the cytotoxic effects of 7-ketocholesterol and 7β-hydroxycholesterol, natural molecules and oils, often associated with the Mediterranean diet, as well as synthetic molecules, have proved effective in vitro. Bioremediation approaches and the use of functionalized nanoparticles are also promising. At the moment, invertebrate and vertebrate models are mainly used to evaluate the metabolism and the toxicity of 7-ketocholesterol and 7β-hydroxycholesterol. The most frequently used models are mice, rats and rabbits. In order to cope with the difficulty of transferring the results obtained in animals to humans, the development of in vitro alternative methods such as organ / body-on-a-chip based on microfluidic technology are hopeful integrative approaches.
... With 7β-OHC, the concentration of 50 μM corresponds to the 50% inhibiting concentration on 158 N cells ( Supplementary Fig. 1). In vitro, it is important to underline that only a part of oxysterols (1-35% depending on the cell type considered) accumulates in the cells [3,31]. It has been established that the median level of 7β-OHC was 1.11 μg/mL (2.8 μM) and 1.43 μg/mL (3.6 μM) in the cerebrospinal fluid of normal subjects and of patients with multiple sclerosis, respectively [9]. ...
... Total lipid was extracted by the Folch's method [37], and fatty acids, sterols and organic acids were quantified as described previously [31,39,40]. Gas chromatography-mass spectrometry (GC-MS) analysis was performed on a Clarus 600D (Perkin Elmer, USA). ...
... The level of MDA was evaluated as previously described [47]. The experimental procedures on 158 N cells were performed as described previously [31]. ...
Article
Oxidative stress and mitochondrial dysfunction contribute to the pathogenesis of neurodegenerative diseases and favor lipid peroxidation, leading to increased levels of 7β-hydroxycholesterol (7β-OHC) which induces oxiapoptophagy (OXIdative stress, APOPTOsis, autoPHAGY). The cytoprotective effects of dimethylfumarate (DMF), used in the treatment of relapsing remitting multiple sclerosis and of monomethylfumarate (MMF), its main metabolite, were evaluated on murine oligodendrocytes 158 N exposed to 7β-OHC (50 μM, 24 h) with or without DMF or MMF (25 μM). The activity of 7β-OHC in the presence or absence DMF or MMF was evaluated on several parameters: cell adhesion; plasma membrane integrity measured with propidium iodide (PI), trypan blue and fluoresceine diacetate (FDA) assays; LDH activity; antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)); generation of lipid peroxidation products (malondialdehyde (MDA), conjugated dienes (CDs)) and protein oxidation products (carbonylated proteins (CPs)); reactive oxygen species (ROS) overproduction conducted with DHE and DHR123. The effect on mitochondria was determined with complementary criteria: measurement of succinate dehydrogenase activity, evaluation of mitochondrial potential (ΔΨm) and mitochondrial superoxide anions (O2●-) production using DiOC6(3) and MitoSOX, respectively; quantification of mitochondrial mass with Mitotracker Red, and of cardiolipins and organic acids. The effects on mitochondrial and peroxisomal ultrastructure were determined by transmission electron microscopy. Intracellular sterol and fatty acid profiles were determined. Apoptosis and autophagy were characterized by staining with Hoechst 33,342, Giemsa and acridine orange, and with antibodies raised against caspase-3 and LC3. DMF and MMF attenuate 7β-OHC-induced cytotoxicity: cell growth inhibition; decreased cell viability; mitochondrial dysfunction (decrease of succinate dehydrogenase activity, loss of ΔΨm, increase of mitochondrial O2●- production, alteration of the tricarboxilic acid (TCA) cycle, and cardiolipins content); oxidative stress induction (ROS overproduction, alteration of GPx, CAT, and SOD activities, increased levels of MDA, CDs, and CPs); changes in fatty acid and cholesterol metabolism; and cell death induction (caspase-3 cleavage, activation of LC3-I in LC3-II). Ultrastructural alterations of mitochondria and peroxisomes were prevented. These results demonstrate that DMF and MMF prevent major dysfunctions associated with neurodegenerative diseases: oxidative stress, mitochondrial dysfunction, apoptosis and autophagy.
... Vitamin B7 (biotin) attenuates 7b-hydroxycholesterol (a lipid peroxidation product) induced cytotoxicity and reported to increase antioxidant activities in neurodegenerative diseases (Sghaier et al., 2019). In the same neurodegenerative model, biotin also restricted the overproduction of O 2 ...
... •2 and H 2 O 2 and consequently, lipid peroxidation and protein carbonylation. Moreover, biotin also improves the mitochondrial function through the upregulation of the activity of SDH and counteracts the loss of transmembrane mitochondrial potential and reduces the overproduction of mitochondrial O 2 •2 (Sghaier et al., 2019). Biotin was reported to induce the endogenous antioxidant response of nuclear factor erythroid 2Àrelated factor 2 (Nrf-2) and limit the cellular ROS as well as mitochondrial ROS production (Fourcade et al., 2020). ...
Chapter
Being the water soluble vitamins, B vitamins largely serve as cofactors or coenzymes for mitochondrial enzymes essential for cell function. This chapter explored the role B vitamins in mitochondrial function and intermediary metabolism as well as impact of B vitamin deficiency on cellular and molecular outcomes in mitochondria. B Vitamins have lately been discovered to be powerful modulators of energy metabolism, particularly mitochondrial activity. Comprehensive account of the role of B vitamins that are relevant to mitochondrial activity and energy metabolism has been discussed. Mitochondria house a diverse range of metabolites required for appropriate cellular growth and development, including amino acid, purine and fatty acid metabolism, as well as the oxidation-reduction reaction. Further, mitochondria are the primary source of reactive oxygen species in cells, and the majority of mitochondrial ROS is produced at the electron transport chain. In this conjuncture, B-vitamins are of critical importance for protection of mitochondrial toxicity and oxidative stress. Moreover, mitochondria are considered as signaling organelles that control a wide range of cellular processes and can even determine the fate of cells. Mitochondria generate a wide range of metabolites that have functions both within the mitochondria and as signaling molecules to other regions of the cell. Current state of knowledge in this aspect has also been explored in this chapter. Overall, B vitamins are required for proper mitochondrial and cellular function, and they constitute a complex web of interrelated processes.
... In 158N, BV-2 and/or N2a cells, the oxysterols (7KC, 7β-OHC, and 24S-OHC) trigger an overproduction of ROS revealed by staining with dihydroethidine/dihydroethidium (DHE), dihydrorhodamine 123 (DHR123) and/or MitoSOX which could trigger an enhanced activity of glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase as well as lipid peroxidation and protein carbonylation, a loss of mitochondrial membrane potential (ΔΨm) measured with DiOC 6 (3), 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. They also favour the formation of large cytoplasmic vacuoles revealed by staining with monodansylcadaverine, considered as autophagic vacuoles and the activation of LC3-I into LC3-II considered as autophagic criteria connected with the autophagosome formation Sghaier, Zarrouk, et al., 2019). In BV-2 cells treated with 7KC, an increase in p62 levels was also reported . ...
... Data obtained from murine nerve cells (oligodendrocytes 158N, microglial BV-2 cells, and neuroblastoma N2a cells) are summarized. In 158N, BV-2 and N2a cells, under treatment with 7KC, 7β-OHC and 24S-OHC, oxidative stress is characterized by an overproduction of ROS (e) revealed after staining with DHE, DHR123 and MitoSOX (detection of ROS overproduction at the mitochondrial level) Nury et al., 2018;Yammine et al., 2020), which is considered to contribute to lipid peroxidation leading to enhanced levels of conjugated dienes (c); in turn, ROS overproduction could trigger an enhanced activity of GPx, SOD and catalase (a, b, d) Sghaier, Zarrouk, et al., 2019); in N2a cells, under treatment with 7KC, compared to untreated cells (control), lower mRNA levels of GPx1 were observed whereas higher levels of SOD1 and SOD2 were detected (f). Whatever the cell type considered (158N, BV-2, and N2a cells), a mode of cell death by apoptosis was observed under treatment with 7KC, 7β-OHC and 24S-OHC; it was characterized by a loss of ΔΨm (g: increased % of DiOC 6 (3) negative cells); by a modification of the subcellular distribution of the mitochondria (h: clusters of mitochondria were often observed in oxysterols-treated cells); by a condensation and/or fragmentation of the nuclei (i) which is characteristic of apoptotic cells; by an internucleosomal DNA fragmentation revealed by the TUNEL assay (Ragot et al., 2013) (j); and by caspase-3 activation, PARP degradation and lower Bcl-2 level (k); these characteristics support an activation of the mitochondrial pathway during oxysterol-induced apoptosis. ...
Article
Oxysterols are oxidized forms of cholesterol generated from cholesterol either by auto-oxidation, enzymatic processes or both. Some of them (7-ketocholesterol, 7β-hydroxycholesterol, 24(S)-hydroxycholesterol), when used at cytotoxic concentrations on different cell types from different species (mesenchymal bone marrow cells, monocytic cells, nerve cells), induce a type of cell death associated with OXIdative stress and several characteristics of APOPTOsis and autoPHAGY, defined as oxiapoptophagy. Oxidative stress is associated with overproduction of reactive oxygen species, increased antioxidant enzyme activities, lipid peroxidation and protein carbonylation. Apoptosis is associated with activation of the mitochondrial pathway: opening of the mitochondrial permeability pore, loss of mitochondrial membrane potential, caspase-3 activation, poly (ADP-ribose) polymerase (PARP) degradation, nuclear condensation and/or fragmentation. Autophagy is characterized by autophagic vacuoles revealed by monodansylcadaverine staining and transmission electron microscopy, and an increased ratio (LC-3II/LC-3I). In addition, morphological, topographical and functional changes of the peroxisome are observed.
... Biotin (vitamin B7 or vitamin H) is involved in utilization of vital structural molecules of the tissues (lipids, sugars, proteins), and its supplementation may account for decrease in LP and improved mitochondrial function, improving their radical formation potential [134]. High doses of biotin supplementation (median = 300 mg/day, divided in three doses, almost 10,000 times the adequate daily intake) had positive results in more than 90% of patients with SPMS and PPMS, and the clinical effects were obvious after two or even eight months after the onset of treatment [135]. ...
Article
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Multiple sclerosis (MS) is a complex neurodegenerative disease. Although its pathogenesis is rather vague in some aspects, it is well known to be an inflammatory process characterized by inflammatory cytokine release and oxidative burden, resulting in demyelination and reduced remyelination and axonal survival together with microglial activation. Antioxidant compounds are gaining interest towards the manipulation of MS, since they offer, in most of the cases, many ben-efits, due to their pleiotropical activity, that mainly derives from the oxidative stress decrease. This review analyzes research articles, of the last decade, which describe biological in vitro, in vivo and clinical evaluation of various categories of the most therapeutically applied natural antioxidant compounds, and some of their derivatives, with anti-MS activity. It also summarizes some of the main characteristics of MS and the role the reactive oxygen and nitrogen species may have in its progression, as well as their relation with the other mechanistic aspects of the disease, in order for the multi-targeting potential of those antioxidants to be defined and the source of origination of such activity explained. Antioxidant compounds with specific characteristics are expected to affect positively some aspects of the disease, and their potential may render them as effective candidates for neurological impairment reduction in combination with the MS treatment regimen. However, more studies are needed in order such antioxidants to be established as recommended treatment to MS patients.
... This requires further investigations to examine these mechanisms. These findings are in agreement with previous reports highlighting the cytotoxicity of oxysterols in other cell types [27,28]. Indeed, the phosphorylated Akt/total Akt ratio was reduced in 7βOH-treated monocytes [18]. ...
Article
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Exercise induces cardioprotection against myocardial infarction, despite obesity, by restoring pro-survival pathways and increasing resistance of mitochondrial permeability transition pore (mPTP) opening at reperfusion. Among the mechanisms involved in the inactivation of these pathways, oxysterols appear interesting. Thus, we investigated the influence of regular exercise on the reperfusion injury salvage kinase (RISK) pathway, oxysterols, and mitochondria, in the absence of ischemia-reperfusion. We also studied 7β-hydroxycholesterol (7βOH) concentration (mass spectrometry) in human lean and obese subjects. Wild-type (WT) and obese (ob/ob) mice were assigned to sedentary conditions or regular treadmill exercise. Exercise significantly increased Akt phosphorylation, whereas 7βOH concentration was reduced. Moreover, exercise induced the translocation of PKCε from the cytosol to mitochondria. However, exercise did not affect the calcium concentration required to open mPTP in the mitochondria, neither in WT nor in ob/ob animals. Finally, human plasma 7βOH concentration was consistent with observations made in mice. In conclusion, regular exercise enhanced the RISK pathway by increasing kinase phosphorylation and PKCε translocation and decreasing 7βOH concentration. This activation needs the combination with stress conditions, i.e., ischemia-reperfusion, in order to inhibit mPTP opening at the onset of reperfusion. The human findings suggest 7βOH as a candidate marker for evaluating cardiovascular risk factors in obesity.
... Biotin is an essential cofactor for carboxylases to control energy metabolism and redox balance (118). In X-ALD mice models, treatment with a high dosage of biotin removes excessive ROS by inducing the endogenous antioxidant response of NRF2 and normalizes redox in vivo via recovering mitochondrial biogenesis and energy supplement to improve symptoms of dyskinesia and axon injury consequently. ...
Article
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X-linked adrenoleukodystrophy (X-ALD) is an inherited disease caused by a mutation in the ABCD1 gene encoding a peroxisomal transmembrane protein. It is characterized by the accumulation of very-long-chain fatty acids (VLCFAs) in body fluids and tissues, leading to progressive demyelination and adrenal insufficiency. ALD has various phenotypes, among which the most common and severe is childhood cerebral adrenoleukodystrophy (CCALD). The pathophysiological mechanisms of ALD remain unclear, but some in vitro/in vivo research showed that VLCFA could induce oxidative stress and inflammation, leading to damage. In addition, the evidence that oxidative stress and inflammation are increased in patients with X-ALD also proves that it is a potential mechanism of brain and adrenal damage. Therefore, normalizing the redox balance becomes a critical therapeutic target. This study focuses on the possible predictors of the severity and progression of X-ALD, the potential mechanisms of pathogenesis, and the promising targeted drugs involved in oxidative stress and inflammation.
... Besides apoptosis, an autophagic cell death was reported in various cell types treated with oxysterols [29]. The generation of ROS induces a protective mechanism of autophagy in MM cells and if the unfolded protein response is chronically compromised, autophagy results in cell death [30]. ...
Article
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Multiple myeloma (MM) is an incurable plasma cell malignancy with frequent patient relapse due to innate or acquired drug resistance. Cholesterol metabolism is reported to be altered in MM; therefore, we investigated the potential anti-myeloma activity of two cholesterol derivatives: the 5,6 α- and 5,6 β-epoxycholesterol (EC) isomers. To this end, viability assays were used, and isomers were shown to exhibit important anti-tumor activity in vitro in JJN3 and U266 human myeloma cell lines (HMCLs) and ex vivo in myeloma patients’ sorted CD138+ malignant cells. Moreover, we confirmed that 5,6 α-EC and 5,6 β-EC induced oxiapoptophagy through concomitant oxidative stress and caspase-3-mediated apoptosis and autophagy. Interestingly, in combination treatment a synergistic interaction was observed between 5,6 α-EC and 5,6 β-EC on myeloma cells. These data highlight a striking anti-tumor activity of 5,6 α-EC and 5,6 β-EC bioactive molecules against human myeloma cells, paving the way for their potential role in future therapeutic strategies in MM.
... In additional, we detected the ROS by flow cytometry and found that GEP could decrease the expression of ROS, suggesting that apoptosis of ICCs was reduced by GEP on the basis of lowering the ROS. This point coincides with the previous study [37]. ...
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Background. Gastric electrical pacing (GEP) could restore interstitial cells of Cajal in diabetic rats. M2 macrophages contribute to the repair of interstitial cells of Cajal injury though secreting heme oxygenase-1 (HO-1). The aim of the study is to investigate the effects and mechanisms of gastric electrical pacing on M2 macrophages in diabetic models. Methods. Sixty male Sprague-Dawley rats were randomized into control, diabetic (DM), diabetic with the sham GEP (DM+SGEP), diabetic with GEP1 (5.5 cpm, 100 ms, 4 mA) (DM+GEP1), diabetic with GEP2 (5.5 cpm, 300 ms, 4 mA) (DM+GEP2), and diabetic with GEP3 (5.5 cpm, 550 ms, 4 mA) (DM+GEP3) groups. The apoptosis of interstitial cells of Cajal and the expression of macrophages were detected by immunofluorescence technique. The expression levels of the Nrf2/HO-1 and NF-κB pathway were evaluated using western blot analysis or immunohistochemical method. Malonaldehyde, superoxide dismutase, and reactive oxygen species were tested to reflect the level of oxidative stress. Results. Apoptosis of interstitial cells of Cajal was increased in the DM group but significantly decreased in the DM+GEP groups. The total number of macrophages was almost the same in each group. In the DM group, M1 macrophages were increased and M2 macrophages were decreased. However, M2 macrophages were dramatically increased and M1 macrophages were reduced in the DM+GEP groups. Gastric electrical pacing improved the Nrf2/HO-1 pathway and downregulated the phosphorylation of NF-κB. In the DM group, the levels of malonaldehyde and reactive oxygen species were elevated and superoxide dismutase was lowered, while gastric electrical pacing reduced the levels of malonaldehyde and reactive oxygen species and improved superoxide dismutase. Conclusion. Gastric electrical pacing reduces apoptosis of interstitial cells of Cajal though promoting M2 macrophages polarization to play an antioxidative stress effect in diabetic rats, which associates with the activated Nrf2/HO-1 pathway and the phosphorylation of NF-κB pathway. 1. Introduction For centuries, gastrointestinal motility disorders, one of the most frequently occurring diseases, have kept perplexed people’s lives with decades of diabetes mellitus [1]. Gastroparesis, namely delayed gastric emptying, is a disorder that slows or reduces the food transit from the stomach to the small intestine without mechanical obstruction. However, effective therapies for gastroparesis remain elusive with limited roles and side effects. Fortunately, nondrug treatments, such as electroacupuncture (EA) and gastric electrical stimulation (GES), are gradually taken seriously because of its apparent efficacy without side effects. In particular, long pulse GES, for it induces slow waves also referred to as gastric electrical pacing (GEP), has a direct impact on gastric motility. At present, GEP develops as an alternative therapy of gastroparesis, but the mechanisms underlying its efficacy remain unclear. Interstitial cells of Cajal (ICCs) serve as pacemakers and generate slow waves spontaneously in the stomach. Defect of ICCs has been consistently found in both humans and animal models with diabetic gastroparesis [2, 3]. We have reported that long-pulse GES could repair the injured ICCs partly by IGF-1 signaling pathway and enhancing the proliferation of ICCs [4, 5]. However, apoptosis of ICCs was also certificated in the stomach of gastroparesis [6]; the effects and mechanisms of GEP on apoptosis of ICCs need to be further clarified. Macrophages have two different phenotypes: proinflammatory M1 macrophages and anti-inflammatory M2 macrophages. They can be transformed into each other in a certain internal environment. Studies have shown that gastrointestinal motility disorder did not occur in diabetic mice with the absence of macrophages, suggesting that macrophages may participate in the development of gastroparesis [7]. It is also reported that phenotypic polarization of M2 macrophages could improve the delayed gastric emptying [8]. Further studies have shown that there was no significant change in the total number of macrophages in animal models and patients of diabetic gastroparesis, while CD206+ M2 macrophages were selectively decreased, accompanied by ICC deletion, resulting in delayed gastric emptying [9–11]. Accordingly, GEP may promote the phenotypic polarization of M2 macrophages to improve ICC expression and gastric emptying. Heme oxygenase-1 (HO-1) is a widely existing antioxidant defense enzyme, which is not expressed or low expressed in normal tissues, but upregulated during stress playing an anti-inflammatory, antioxidative, and antiapoptotic role. In the gastrointestinal tract, HO-1 is mainly produced and expressed by resident M2 macrophages [12]. Recently, in a study of diabetic gastroparesis model, it has been found that the downregulation of HO-1 could not resist oxidative stress injury, which leads to the destruction of ICCs network and delayed gastric emptying, but upregulation of HO-1 could repair the injury of ICCs and improve the delayed gastric emptying [13, 14]. Similarly, Tian LG et al. reported that the expression of HO-1 in gastric antrum of diabetic gastroparesis mice was significantly decreased, and EA could improve the expression of HO-1 and gastric motility [15]. Therefore, we speculate that GEP may play a key role in improving gastric motility disorder by upregulating HO-1 to repair ICC injury. Also, studies have shown that HO-1 can protect and reverse oxidative stress damage to ICCs [13]. Stem cell factor (also known as SCF, c-kit ligand) is essential for survival and maintenance of ICCs, but there are few reports about the correlation between HO-1 and SCF. Recent studies [16] have shown that the NF-κB signaling pathway was activated in diabetic gastrointestinal motility disorders and the decrease expression of SCF/c-kit causes the increase of ICC apoptosis, suggesting that the activation of NF-κB signaling may be an important factor in ICC apoptosis. It is also reported that nuclear factor erythroid 2-related factor 2 (Nrf2) plays a critical role in defending against inflammation in different tissues via activation of phase II enzyme HO-1 and inhibition of the NF-κB signaling pathway [17]. Other studies [18] have shown that HO-1 can inhibit the phosphorylation of NF-κB p65, promote the binding of anti-apoptotic genes to NF-κB, and promote gene transcription to play an antiapoptotic role. In addition, studies in human lung fibroblasts have shown that nuclear transcription factor NF-κB binds to enhancers of SCF gene and promotes SCF transcription [19]. We assumed that HO-1 may play an antiapoptotic role by inhibiting the phosphorylation of NF-κB p65. In this study, we aimed to explore the effects of GEP on ICC apoptosis and phenotypic polarization of macrophages and to investigate the possible mechanisms of renovation of ICC injury in diabetic rats. 2. Materials and Methods 2.1. Animals Male Sprague-Dawley rats (weighing 160-200 g, ) were purchased from Jinan Pengyue Experimental Animal Breeding Co. Ltd. (Shandong, China) and were kept in the suitable laboratory conditions (22-23°C, 12/12 h light-dark cycle) with food and water ad libitum. The experimental procedures were implemented, following the ethical guidelines from the Animal Care and Use Committee of Binzhou Medical University Hospital Laboratory Animal Ethical Committee. The rats were randomly divided into the normal control, diabetes (DM), diabetic+sham GEP (DM+SGEP), and diabetic+GEP (DM+GEP) groups (Figure 1).
... 12 Oxidative stress, which is understood as an imbalance between pro-oxidant and antioxidant molecules, is associated with schizophrenia progression, as well as with the development of other neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. 11,[13][14][15] In this case, progression is stimulated by an increase in reactive oxygen species (ROS) production, decreased antioxidant defense, or a combination of the two. 13 The increase in ROS production may occur in the brain as a consequence of high oxygen saturation resulting from intense neuronal metabolic activity that damages the tissues, lipid membranes, proteins, enzymes, and DNA. ...
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... Any mitochondrial dysfunction impacts cellular metabolism and negatively affects the health of the organism. In particular mitochondrial dysfunction, at the level of neurons, involves oxidative damage that can lead to cell death generating neurodegeneration [29]. In these cases, supplementation with antioxidant compounds, of an exogenous or endogenous nature, has been shown to reduce the severity of many neurodegenerative diseases [30]. ...
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Thesis
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Oxysterols are cholesterol metabolites that can be produced through enzymatic or radical processes. They constitute a large family of lipids (i.e. the oxysterome) involved in a plethora of physiological processes. They can act through GPCR (e.g. EBI2, SMO, CXCR2), nuclear receptors (LXR, ROR, ER?) and through transporters or regulatory proteins. Their physiological effects encompass cholesterol, lipid and glucose homeostasis. Additionally, they were shown to be involved in other processes such as immune regulatory functions and brain homeostasis. First studied as precursors of bile acids, they quickly emerged as interesting lipid mediators. Their levels are greatly altered in several pathologies and some oxysterols (e.g. 4?-hydroxycholesterol or 7?-hydroxycholestenone) are used as biomarkers of specific pathologies. In this review, we discuss the complex metabolism and molecular targets (including their binding properties) of these bioactive lipids in human and mice. We also discuss the genetic mouse models currently available to interrogate their effects in pathophysiological settings. We also summarize the levels of oxysterols reported in two key organs in oxysterol metabolism (liver and brain), plasma and cerebrospinal fluid. Finally, we consider future opportunities and directions in the oxysterol field in order to gain a better insight and understanding of the complex oxysterol system.
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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.
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Introduction: Multiple sclerosis (MS) is a chronic, potentially highly disabling neurological disorder. No disease-modifying treatments are approved in the progressive and not active forms of the disease. Areas covered: High doses of biotin were tested in an open-label pilot study involving 23 patients with progressive MS and reported positive results. A randomized, double-blind, placebo-controlled trial in 154 progressive MS patients confirmed the beneficial effect of MD1003 (high-dose biotin) on reversing or stabilizing disability progression, with a good safety profile. It is proposed that MD1003 in progressive MS 1) increases energy production in demyelinated axons and/or 2) enhances myelin synthesis in oligodendrocytes. Biotin is highly bioavailable; absorption and excretion are rapid. The major route of elimination is urinary excretion. Expert opinion: A high oral dose of biotin seems generally well tolerated but a few important safety concerns were identified: 1) teratogenicity in one species and 2) interference with some biotin-based laboratory immunoassays. The animal toxicity data are limited at such high doses. Further preclinical studies would be useful to address the mechanism of action of MD1003. Assessment of clinical benefit duration in responders will be also very important to set. Results of randomized, placebo-controlled trial are reassuring and provide hope for the treatment of progressive MS.
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Oxidative injury plays a major role in brain damage in many age-related human brain diseases and is particularly pronounced in the progressive stage of multiple sclerosis. In the latter it is related to the chronic inflammatory process and is amplified by brain changes due to aging and accumulation of disease burden. It induces demyelination and neurodegeneration by direct oxidation of lipids, proteins and DNA as well as by the induction of mitochondrial injury, which results in energy deficiency and further amplification of oxygen radical production. It affects neurons and all types of glia cells, but neurons and oligodendrocytes are most vulnerable. Difference in the susceptibility for oxidative injury between different cellular components of the central nervous system appears to be due to cell type specific differences in anti-oxidant defense mechanisms, iron loading, cellular susceptibility to apoptosis induction and energy demand.
Article
AimsCholesterol plays an essential role in membrane structure and function, being especially important in the brain. Alteration of brain cholesterol synthesis and metabolism has been demonstrated in several Huntington's disease (HD) mouse and cell models, however less is known about these alterations in human tissue. This study aimed to identify alterations to cholesterol synthetic and metabolic pathways in human HD brain tissue.MethodsA broad range of cholesterol synthetic precursors, metabolites and oxidation products were measured by GC-MS/MS in five regions of human post-mortem HD brain and compared to age- and sex-matched control tissues. The level of enzymes that regulate cholesterol homeostasis, cholesterol 24-hydroxylase and delta(24)-sterol reductase were investigated by Western blotting and qPCR in putamen.ResultsThe most significant changes were localised to the putamen, where a 60% decrease in 24(S)-hydroxycholesterol, 30% increase in cholesterol and 100 to 200% increase in synthetic precursors (lathosterol, zymosterol and desmosterol) was detected. The enzymes cholesterol 24-hydroxylase and delta(24)-sterol reductase were also significantly decreased in HD putamen as compared to control tissues. Free radical-generated cholesterol oxidation products 7-keto cholesterol and 7β-hydroxycholesterol were also increased by 50 to 70% in HD putamen.Conclusion Human HD brain has significantly decreased cholesterol metabolism and disrupted cholesterol homeostasis. Our data also indicates that lipid oxidative stress accompanies HD pathology.
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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 beta-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.
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Mitochondrial dysfunction has been implicated in the development and progression of multiple sclerosis (MS) lesions. Mitochondrial alterations might occur as a response to demyelination and inflammation, since demyelination leads to an increased energy demand in axons and could thereby affect the number, distribution and activity of mitochondria. We have studied the expression of mitochondrial proteins and mitochondrial enzyme activity in active demyelinating and chronic inactive MS lesions. Mitochondrial protein expression and enzyme activity in active and chronic inactive MS lesions was investigated using (immuno)histochemical and biochemical techniques. The number of mitochondria and their co-localization with axons and astrocytes within MS lesions and adjacent normal-appearing white matter (NAWM) was quantitatively assessed. In both active and inactive lesions we observed an increase in mitochondrial protein expression as well as a significant increase in the number of mitochondria. Mitochondrial density in axons and astrocytes was significantly enhanced in active lesions compared to adjacent NAWM, whereas a trend was observed in inactive lesions. Complex IV activity was strikingly up-regulated in MS lesions compared to control white matter and, to a lesser extent, NAWM. Finally, we demonstrated increased immunoreactivity of the mitochondrial stress protein mtHSP70 in MS lesions, particularly in astrocytes and axons. Our data indicate the occurrence of severe mitochondrial alterations in MS lesions, which coincides with enhanced mitochondrial oxidative stress. Together, these findings support a mechanism whereby enhanced density of mitochondria in MS lesions might contribute to the formation of free radicals and subsequent tissue damage.
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