Search

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.

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 &micro, M, 48 h) induces cytotoxic effects characterized by an induction of cell death. When associated with RSV, QCT and API (3.125, 6.25 &micro, M), 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 (&Psi, &Delta, m) and attenuating the decreased gene expression and/or protein level of AMP-activated protein kinase &alpha, (AMPK&alpha, ), sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor &gamma, coactivator-1&alpha, (PGC-1&alpha, ) 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 &beta, oxidation (Abcd1, Acox1, Mfp2, Thiolase A). The 7KC-induced decrease in ABCD1 and multifunctional enzyme type 2 (MFP2), two proteins involved in peroxisomal &beta, 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&ndash, 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.

Oxysterols are oxidized forms of cholesterol generated from cholesterol by auto-oxidation, enzymatic processes, or both. Some of them (7-ketocholesterol, 7β-hydroxycholesterol and 24(S)-hydroxycholesterol), when used at cytotoxic concentrations on different cell types from different species (mesenchymal bone marrow cells, monocytic cells and 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 ROS, 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, PARP degradation, nuclear condensation and/or fragmentation. Autophagy is characterized by autophagic vacuoles revealed by monodansylcadaverine staining and transmission electron microscopy, plus increased ratio of LC-3II/LC-3I. In addition, morphological, topographical and functional changes of the peroxisome are observed. LINKED ARTICLES: This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.

International audience; 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 7beta-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 7beta-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 7beta-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.

Peroxisomopathies are qualitative or quantitative deficiencies in peroxisomes which lead to increases in the level of very-long-chain fatty acids (VLCFA) and can be associated with more or less pronounced dysfunction of central nervous system cells: glial and microglial cells. Currently, in frequent neurodegenerative diseases, Alzheimer’s disease (AD) and multiple sclerosis (MS), peroxisomal dysfunction is also suspected due to an increase in VLCFA, which can be associated with a decrease of plasmalogens, in these patients. Moreover, in patients suffering from peroxisomopathies, such as X-linked adrenoleukodystrophy (X-ALD), AD, or MS, the increase in oxidative stress observed leads to the formation of cytotoxic oxysterols: 7-ketocholesterol (7KC) and 7β-hydroxycholesterol (7β-OHC). These observations led to the demonstration that 7KC and 7β-OHC alter the biogenesis and activity of peroxisomes in glial and microglial cells. In X-ALD, AD, and MS, it is suggested that 7KC and 7β-OHC affecting the peroxisome, and which also induce mitochondrial dysfunctions, oxidative stress, and inflammation, could promote neurodegeneration. Consequently, the study of oxisome in peroxisomopathies, AD and MS, could help to better understand the pathophysiology of these diseases to identify therapeutic targets for effective treatments.

Cholesterol oxidation products, also named oxysterols, can be formed either by cholesterol auto-oxidation, enzymatically or both. Among these oxysterols, 7-ketocholesterol (7KC) is mainly formed during radical attacks that take place on the carbon 7 of cholesterol. As increased levels of 7KC have been found in the tissues, plasma and/or cerebrospinal fluid of patients with major diseases, especially age-related diseases (cardiovascular diseases, eye diseases, neurodegenerative diseases), some cancers, and chronic inflammatory diseases, it is suspected that 7KC, could contribute to their development. Since 7KC, provided by the diet or endogenously formed, is not or little efficiently metabolized, except in hepatic cells, its cellular accumulation can trigger numerous side effects including oxidative stress, inflammation and cell death. To counteract 7KC-induced side effects, it is necessary to characterize the metabolic pathways activated by this oxysterol to identify potential targets for cytoprotection and geroprotection. Currently, several natural compounds (tocopherols, fatty acids, polyphenols, etc) or mixtures of compounds (oils) used in traditional medicine are able to inhibit the deleterious effects of 7KC. The different molecules identified could be valued in different ways (functional foods, recombinant molecules, theranostic) to prevent or treat diseases associated with 7KC.

The brain, which is a cholesterol-rich organ, can be subject to oxidative stress in a variety of pathophysiological conditions, age-related diseases and some rare pathologies. This can lead to the formation of 7-ketocholesterol (7KC), a toxic derivative of cholesterol mainly produced by auto-oxidation. So, preventing the neuronal toxicity of 7KC is an important issue to avoid brain damage. As there are numerous data in favor of the prevention of neurodegeneration by the Mediterranean diet, this study aimed to evaluate the potential of a series of polyphenols (resveratrol, RSV, quercetin, QCT, and apigenin, API) as well as &omega, 3 and &omega, 9 unsaturated fatty acids (&alpha, linolenic acid, ALA, eicosapentaenoic acid, EPA, docosahexaenoic acid, DHA, and oleic acid, OA) widely present in this diet, to prevent 7KC (50 &micro, M)-induced dysfunction of N2a neuronal cells. When polyphenols and fatty acids were used at non-toxic concentrations (polyphenols: &le, 6.25 &micro, M, fatty acids: &le, 25 &micro, M) as defined by the fluorescein diacetate assay, they greatly reduce 7KC-induced toxicity. The cytoprotective effects observed with polyphenols and fatty acids were comparable to those of &alpha, tocopherol (400 &micro, M) used as a reference. These polyphenols and fatty acids attenuate the overproduction of reactive oxygen species and the 7KC-induced drop in mitochondrial transmembrane potential (&Delta, &Psi, m) measured by flow cytometry after dihydroethidium and DiOC6(3) staining, respectively. Moreover, the studied polyphenols and fatty acids reduced plasma membrane permeability considered as a criterion for cell death measured by flow cytometry after propidium iodide staining. Our data show that polyphenols (RSV, QCT and API) as well as &omega, 9 unsaturated fatty acids (ALA, EPA, DHA and OA) are potent cytoprotective agents against 7KC-induced neurotoxicity in N2a cells. Their cytoprotective effects could partly explain the benefits of the Mediterranean diet on human health, particularly in the prevention of neurodegenerative diseases.