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The Mediterranean diet is associated with health benefits due to bioactive compounds such as polyphenols. The biological activities of three polyphenols (quercetin (QCT), resveratrol (RSV), apigenin (API)) were evaluated in mouse neuronal N2a cells in the presence of 7-ketocholesterol (7KC), a major cholesterol oxidation product increased in patients with age-related diseases, including neurodegenerative disorders. In N2a cells, 7KC (50 µM; 48 h) induces cytotoxic effects characterized by an induction of cell death. When associated with RSV, QCT and API (3.125; 6.25 µM), 7KC-induced toxicity was reduced. The ability of QCT, RSV and API to prevent 7KC-induced oxidative stress was characterized by a decrease in reactive oxygen species (ROS) production in whole cells and at the mitochondrial level; by an attenuation of the increase in the level and activity of catalase; by attenuating the decrease in the expression, level and activity of glutathione peroxidase 1 (GPx1); by normalizing the expression, level and activity of superoxide dismutases 1 and 2 (SOD1, SOD2); and by reducing the decrease in the expression of nuclear erythroid 2-like factor 2 (Nrf2) which regulates antioxidant genes. QCT, RSV and API also prevented mitochondrial dysfunction in 7KC-treated cells by counteracting the loss of mitochondrial membrane potential (ΨΔm) and attenuating the decreased gene expression and/or protein level of AMP-activated protein kinase α (AMPKα), sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) implicated in mitochondrial biogenesis. At the peroxisomal level, QCT, RSV and API prevented the impact of 7KC by counteracting the decrease in ATP binding cassette subfamily D member (ABCD)3 (a peroxisomal mass marker) at the protein and mRNA levels, as well as the decreased expresssion of genes associated with peroxisomal biogenesis ( Pex13 , Pex14 ) and peroxisomal β-oxidation ( Abcd1 , Acox1 , Mfp2 , Thiolase A ). The 7KC-induced decrease in ABCD1 and multifunctional enzyme type 2 (MFP2), two proteins involved in peroxisomal β-oxidation, was also attenuated by RSV, QCT and API. 7KC-induced cell death, which has characteristics of apoptosis (cells with fragmented and/or condensed nuclei; cleaved caspase-3; Poly(ADP-ribose) polymerase (PARP) fragmentation) and autophagy (cells with monodansyl cadaverine positive vacuoles; activation of microtubule associated protein 1 light chain 3-I (LC3-I) to LC3-II, was also strongly attenuated by RSV, QCT and API. Thus, in N2a cells, 7KC induces a mode of cell death by oxiapoptophagy, including criteria of OXIdative stress, APOPTOsis and autoPHAGY, associated with mitochondrial and peroxisomal dysfunction, which is counteracted by RSV, QCT, and API reinforcing the interest for these polyphenols in prevention of diseases associated with increased 7KC levels.

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 7β-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-5β-cholane (compound 1a), and 23-(4-methylfuran-2,5-dione)-3α,7α-dihydroxy-24-nor-5β-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 7β-hydroxycholesterol. The cell clonogenic survival assay showed that compound 1b was more cytotoxic than compound 1a and 7β-hydroxycholesterol. Compound 1b and 7β-hydroxycholesterol also induce cell cycle modifications. In addition, compounds 1a and 1b, and 7β-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 7β-hydroxycholesterol-induced cell death, respectively, supporting that 7β-hydroxycholesterol induces survival autophagy. Alpha-tocopherol also strongly attenuates 7β-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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In recent years, a particular interest has focused on the accumulation of fatty acids with very long chains (VLCFA) in the occurrence of neurodegenerative diseases such as Alzheimer's disease, multiple sclerosis or dementia. Indeed, it seems increasingly clear that this accumulation of VLCFA in the central nervous system is accompanied by a progressive demyelination resulting in death of neuronal cells. Nevertheless, molecular mechanisms by which VLCFA result in toxicity remain unclear. This study highlights for the first time in 3 different cellular models (oligodendrocytes 158 N, primary mouse brain culture, and patient fibroblasts) the types of cell death involved where VLCFA-induced ROS production leads to autophagy. The autophagic process protects the cell from this VLCFA-induced toxicity. Thus, autophagy in addition to oxidative stress can offer new therapeutic approaches., (Copyright © 2019. Published by Elsevier Inc.)

Context: Mints (Lamiaceae) are used as traditional remedies for the treatment of several diseases. Their extracts are recognized as anti-inflammatory compounds., Objective: This study characterized the cytotoxic effects of Mentha spicata L. (MS), Mentha pulegium L. (MP) and Mentha rotundifolia (L). Huds (MR) on macrophage cells (RAW264.7; U937) and determined their impact on apoptosis and autophagy, which can play a role in controlling inflammation., Materials and Methods: The extracts were prepared in culture medium and tested from 25 to 400 μg/mL after 24-48 h of treatment. To show the effect of the aqueous ethanol (50%) extracts on apoptosis and authophagy, the presence of cleaved caspase-3, and the conversion of LC3-I to LC3-II was evaluated by Western blotting., Results: Compared with the MTT assay, crystal violet showed a pronounced decrease in the number of cells with all extracts at 48 h. Calculated IC 50 values were 257.31, 207.82 and 368.02 μg/mL for MS, MP and MR, respectively. A significant increase in PI positive cells was observed with all extracts at 200-400 μg/mL. Mitochondrial dysfunctions and nuclear morphological changes were detected with MS and MR extracts at 400 μg/mL. At this concentration, no cleaved caspase-3 was found whereas stabilized caspase-3 in its dimeric form was identified. MS and MR extracts also favour LC3-I to LC3-II conversion which is a criterion of autophagy., Conclusions: The cytotoxic profiles depend on the extracts considered; MS extract showed the strong activity. However, all the mint extracts studied interact with the apoptotic and autophagic pathways at elevated concentrations.

Increased levels of 7-ketocholesterol (7KC), which results mainly from cholesterol auto-oxidation, are often found in the plasma and/or cerebrospinal fluid of patients with neurodegenerative diseases and might contribute to activation of microglial cells involved in neurodegeneration. As major cellular dysfunctions are induced by 7KC, it is important to identify molecules able to impair its side effects. Since consumption of olive and argan oils, and fish is important in the Mediterranean diet, the aim of the study was to determine the ability of oleic acid (OA), a major compound of olive and argan oil, and docosahexaenoic acid (DHA) present in fatty fishes, such as sardines, to attenuate 7KC-induced cytotoxic effects. Since elaidic acid (EA), the trans isomer of OA, can be found in hydrogenated cooking oils and fried foods, its effects on 7KC-induced cytotoxicity were also determined. In murine microglial BV-2 cells, 7KC induces cell growth inhibition, mitochondrial dysfunctions, reactive oxygen species overproduction and lipid peroxidation, increased plasma membrane permeability and fluidity, nuclei condensation and/or fragmentation and caspase-3 activation, which are apoptotic characteristics, and an increased LC3-II/LC3-I ratio, which is a criterion of autophagy. 7KC is therefore a potent inducer of oxiapoptophagy (OXIdation+APOPTOsis+autoPHAGY) on BV-2 cells. OA and EA, but not DHA, also favor the accumulation of lipid droplets revealed with Masson's trichrome, Oil Red O, and Nile Red staining. The cytotoxicity of 7KC was strongly attenuated by OA and DHA. Protective effects were also observed with EA. However, 7KC-induced caspase-3 activation was less attenuated with EA. Different effects of OA and EA on autophagy were also observed. In addition, EA (but not OA) increased plasma membrane fluidity, and only OA (but not EA) was able to prevent the 7KC-induced increase in plasma membrane fluidity. Thus, in BV-2 microglial cells, the principal fatty acids of the Mediterranean diet (OA, DHA) were able to attenuate the major toxic effects of 7KC, thus reinforcing the interest of natural compounds present in the Mediterranean diet to prevent the development of neurodegenerative diseases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Mitochondrial dysfunctions and oxidative stress are involved in several non demyelinating or demyelinating neurodegenerative diseases. Some of them, including multiple sclerosis (MS), are associated with lipid peroxidation processes leading to increased levels of 7-ketocholesterol (7KC). So, the eventual protective effect of dimethylfumarate (DMF), which is used for the treatment of MS, was evaluated on 7KC-treated oligodendrocytes, which are myelin synthesizing cells. To this end, murine oligodendrocytes 158N were exposed to 7KC (25, 50μM) for 24h without or with DMF (1, 25, 50μM). The biological activities of DMF associated or not with 7KC were evaluated by phase contrast microscopy, crystal violet and MTT tests. The impact on transmembrane mitochondrial potential (ΔYm), O 2 - and H 2 production, apoptosis and autophagy was measured by microscopical and flow cytometric methods by staining with DiOC 2 production, apoptosis and autophagy was measured by microscopical and flow cytometric methods by staining with DiOC 6 (3), dihydroethidine and dihydrorhodamine 123, Hoechst 33342, and by Western blotting with the use of specific antibodies raised against uncleaved and cleaved caspase-3 and PARP, and LC3-I/II. DMF attenuates the different effects of 7KC, namely: cell growth inhibition and/or loss of cell adhesion, decrease of ΔΨm, O 2 - overproduction, PARP and caspase-3 cleavage, nuclear condensation and fragmentation, and activation of LC3-I into LC3-II. The ability of DMF to attenuate 7KC-induced reactive oxygen species overproduction, apoptosis, and autophagy on oligodendrocytes reinforces the interest for this molecule for the treatment of MS or other demyelinating diseases.2 O 2 overproduction, PARP and caspase-3 cleavage, nuclear condensation and fragmentation, and activation of LC3-I into LC3-II. The ability of DMF to attenuate 7KC-induced reactive oxygen species overproduction, apoptosis, and autophagy on oligodendrocytes reinforces the interest for this molecule for the treatment of MS or other demyelinating diseases., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)