Molecular mechanisms associated with the effects of ODN on astroglial and microglial cells stressed by an oxidative stress : impact on lipid metabolism and cell death

Les maladies neurodégénératives sont caractérisées par un stress oxydatif associé à des dommages mitochondriaux aboutissant à la mort des cellules neuronales. Pour atténuer ces dommages et favoriser la cytoprotection neuronale ainsi que la neurogenèse, des facteurs neurotrophiques naturels de type endogènes (Neuropeptide : octadécaneuropeptide (ODN)) ou exogènes (Polyphénols : resvératrol (RSV) et apigénine (API)) pourraient être utilisés comme agents thérapeutiques permettant de favoriser la différenciation neuronale des cellules souches immatures et pluripotentes. L’ODN est un peptide produit par les astrocytes et connu comme agent neuroprotecteur puissant d’où l’intérêt d’étudier ses effets sur la mobilisation du calcium, sa capacité à protéger les cellules neuronales contre la mort par apoptose générée par le peroxyde d’hydrogène (H2O2) et d’évaluer son pouvoir à stimuler la neurogenèse en favorisant la différenciation neuronale. Les effets des polyphénols (RSV, API), composés majeurs du régime méditerranéen, sur la neurogenèse ont été également évalués.Les propriétés cytoprotectrices et/ou différenciatrices de l’ODN (10-16 -10-8 M) et des polyphénols (RSV : 6.25 -50 µM ; API : 6.25 -50 µM) ont été essentiellement étudiées sur des cellules de neuroblastomes murins N2a mais aussi sur d’autres lignées nerveuses murines (BV-2, C6) et humaines (SK-N-BE ; CCF-STTG1). La cytoprotection a été mesurée par différents tests de viabilité (FDA, MTT, DiOC6(3), iodure de propidium). La différenciation a été évaluée morphologiquement par la présence de neurites (axones et dendrites) visualisés grâce à différentes techniques de microscopie. L’acide rétinoïque (AR : 6.25 -50 µM) a été utilisé comme contrôle positif d’induction de différenciation. Les voies de signalisation impliquées dans la différenciation neuronale ont été caractérisées. Nous avons aussi étudié l’effet de l’ODN (10-14 M, 48 h) sur la morphologie, la topographie et l’activité des mitochondries et des peroxysomes au cours de la différenciation. Ces deux organites sont impliqués dans le métabolisme des lipides (acides gras, cholestérol).Les résultats obtenus montrent que l’ODN est capable de promouvoir la survie des cellules N2a cultivées en condition de stress oxydatif aigue induit par le H2O2. De plus, l’ODN ainsi que les polyphénols RSV et l’API, qui sont dépourvus d’effet cytotoxique intrinsèque stimulent la croissance des neurites indiquant qu’ils exercent des effets neurotrophiques pro-différenciateurs. Cet effet de l’ODN met en jeu l’activation de son récepteur métabotropique associé aux voies de transduction intracellulaire PKA, PKC ainsi que MAPK / ERKs. De plus, l’ODN stimule la biogenèse des mitochondries et des peroxysomes, organites essentiels dans l’activité axonale (transport axonal et renouvellement). L’étude des voies de signalisation démontre que les effets trophiques du RSV et API mettent en jeu l’activation des voies de transduction de la PKC, PKA ainsi que celle des MAPK / ERKs.Sur la base de ces résultats, la libération d’ODN pourrait être un mécanisme endogène de protection en réponse aux attaques oxydatives et au processus de neurodégénérescence, empêchant la mort cellulaire et favorisant la différenciation des cellules neuronales. Nos travaux mettent aussi en évidence pour la première fois que les polyphénols en plus de leur action antioxydante stimulent la formation, la maturation et l’élongation des neurites des cellules N2a non différenciées. L’ensemble de ces travaux indique que le neuropeptide ODN et les polyphénols RSV et API sont de puissants agents neurotrophiques. Ces molécules présentent donc un intérêt pharmacologique en vue de leur utilisation et/ou de leurs analogues synthétiques pour traiter des maladies neurodégénératives en favorisant la neuroprotection, la neuro-réparation et la neurogenèse.Neurodegenerative diseases are characterized by oxidative stress associated with mitochondrial damages leading to neuronal cell death. To mitigate these damages and promote neuronal cytoprotection and neurogenesis, endogenous (Neuropeptide: octadecaneuropeptide (ODN)) or exogenous (Polyphenols: resveratrol (RSV) and apigenin (API)) natural neurotrophic factors could be used as therapeutic agents to promote neuronal differentiation of immature and pluripotent stem cells. ODN is a peptide produced by astrocytes and known as a powerful neuroprotective agent. It is therefore of interest of studying its effects on the mobilization of calcium, its ability to protect neuronal cells against apoptosis death caused by hydrogen peroxide (H2O2) and evaluate its ability to stimulate neurogenesis by promoting neuronal differentiation. The effects of polyphenols (RSV, API), major compounds of the Mediterranean diet, on neurogenesis were also evaluated.The cytoprotective and/or differentiating properties of ODN (10-16 -10-8 M) and polyphenols (RSV: 6.25 -50 μM, API: 6.25 -50 μM) were mainly studied on murine N2a neuroblastoma cells but also on other murine (BV-2, C6) and human (SK-N-BE, CCF-STTG1) nerve lines. Cytoprotection was measured by various viability tests (FDA, MTT, DiOC6(3), propidium iodide). Differentiation was morphologically evaluated by the presence of neurites (axons and dendrites) and visualized by different microscopical techniques. Retinoic acid (RA: 6.25-50 μM) was used as a positive inductor of differentiation. The signaling pathways involved in neuronal differentiation have been characterized. We also studied the effect of ODN (10-14 M, 48 h) on the morphology, topography and activity of mitochondria and peroxisome during differentiation. These two organelles are involved in the metabolism of lipids (fatty acids, cholesterol).The results obtained show that ODN is able to promote the survival of N2a cells cultured under the conditions of acute oxidative stress induced by H2O2. In addition, ODN as well as polyphenols (RSV and API), which lack intrinsic cytotoxic effects, stimulate neurite outgrowth, indicating that they exert pro-differentiating neurotrophic effects. This effect of ODN involves activation of its metabotropic receptor associated with intracellular transduction pathways PKA, PKC and MAPK / ERKs. In addition, ODN stimulates the biogenesis of mitochondria and peroxisomes, essential organelles in axonal activity (axonal transport and renewal). The study of signaling pathways demonstrate that the trophic effects of RSV and API involve the activation of PKC, PKA and MAPK / ERK transduction pathways.Based on these results, the ODN release could be an endogenous protective mechanism in response to oxidative attacks and process of neurodegeneration, preventing cell death and promoting neuronal cell differentiation. Our work also highlights for the first time that polyphenols, in addition to their antioxidant activity, stimulate the formation, maturation and elongation of neurites of undifferentiated N2a cells. All of this work indicates that ODN neuropeptide and polyphenols (RSV and API) are potent neurotrophic agents. These molecules and/or their synthetic analogues may have pharmacological interest for treating neurodegenerative diseases by promoting neuroprotection, neuro-repair and neurogenesis

Biomarkers of Amyotrophic Lateral Sclerosis: Current Status and Interest of Oxysterols and Phytosterols

Amyotrophic lateral sclerosis (ALS) is a non-demyelinating neurodegenerative disease in adults with motor disorders. Two forms exist: a sporadic form (90% of cases) and a family form due to mutations in more than 20 genes including the Superoxide dismutase 1, TAR DNA Binding Protein, Fused in Sarcoma, chromosome 9 open reading frame 72 and VAPB genes. The mechanisms associated with this pathology are beginning to be known: oxidative stress, glutamate excitotoxicity, protein aggregation, reticulum endoplasmic stress, neuroinflammation, alteration of RNA metabolism. In various neurodegenerative diseases, such as Alzheimer’s disease or multiple sclerosis, the involvement of lipids is increasingly suggested based on lipid metabolism modifications. With regard to ALS, research has also focused on the possible involvement of lipids. Lipid involvement was suggested for clinical arguments where changes in cholesterol and LDL/HDL levels were reported with, however, differences in positivity between studies. Since lipids are involved in the membrane structure and certain signaling pathways, it may be considered to look for oxysterols, mainly 25-hydroxycholesterol and its metabolites involved in immune response, or phytosterols to find suitable biomarkers for this pathology

Induction of Neuronal Differentiation of Murine N2a Cells by Two Polyphenols Present in the Mediterranean Diet Mimicking Neurotrophins Activities: Resveratrol and Apigenin

In the prevention of neurodegeneration associated with aging and neurodegenerative diseases (Alzheimer’s disease, Parkinson’s disease), neuronal differentiation is of interest. In this context, neurotrophic factors are a family of peptides capable of promoting the growth, survival, and/or differentiation of both developing and immature neurons. In contrast to these peptidyl compounds, polyphenols are not degraded in the intestinal tract and are able to cross the blood–brain barrier. Consequently, they could potentially be used as therapeutic agents in neurodegenerative pathologies associated with neuronal loss, thus requiring the stimulation of neurogenesis. We therefore studied the ability to induce neuronal differentiation of two major polyphenols present in the Mediterranean diet: resveratrol (RSV), a major compound found in grapes and red wine, and apigenin (API), present in parsley, rosemary, olive oil, and honey. The effects of these compounds (RSV and API: 6.25–50 µM) were studied on murine neuro-2a (N2a) cells after 48 h of treatment without or with 10% fetal bovine serum (FBS). Retinoic acid (RA: 6.25–50 µM) was used as positive control. Neuronal differentiation was morphologically evaluated through the presence of dendrites and axons. Cell growth was determined by cell counting and cell viability by staining with fluorescein diacetate (FDA). Neuronal differentiation was more efficient in the absence of serum than with 10% FBS or 10% delipidized FBS. At concentrations inducing neuronal differentiation, no or slight cytotoxicity was observed with RSV and API, whereas RA was cytotoxic. Without FBS, RSV and API, as well as RA, trigger the neuronal differentiation of N2a cells via signaling pathways simultaneously involving protein kinase A (PKA)/phospholipase C (PLC)/protein kinase C (PKC) and MEK/ERK. With 10% FBS, RSV and RA induce neuronal differentiation via PLC/PKC and PKA/PLC/PKC, respectively. With 10% FBS, PKA and PLC/PKC as well as MEK/ERK signaling pathways were not activated in API-induced neuronal differentiation. In addition, the differentiating effects of RSV and API were not inhibited by cyclo[DLeu5] OP, an antagonist of octadecaneuropeptide (ODN) which is a neurotrophic factor. Moreover, RSV and API do not stimulate the expression of the diazepam-binding inhibitor (DBI), the precursor of ODN. Thus, RSV and API are able to induce neuronal differentiation, ODN and its receptor are not involved in this process, and the activation of the (PLC/PKC) signaling pathway is required, except with apigenin in the presence of 10% FBS. These data show that RSV and API are able to induce neuronal differentiation and therefore mimic neurotrophin activity. Thus, RSV and API could be of interest in regenerative medicine to favor neurogenesis

The effect of oxysterols on nerve impulses.

International audienceThe propagation of nerve impulses in myelinated nerve fibers depends on a number of factors involving the myelin and neural axons. In several neurodegenerative diseases, nerve impulses can be affected by the structural and biochemical characteristics of the myelin sheath and the activity of ion channels located in the nodes of Ranvier. Though it is generally accepted that lipid disorders are involved in the development of neurodegenerative diseases, little is known about their impact on nerve impulses. Cholesterol oxide derivatives (also called oxysterols), which are either formed enzymatically or as a result of cholesterol auto-oxidation or both, are often found in abnormal levels in the brain and body fluids of patients with neurodegenerative diseases. This leads to the question of whether these molecules, which can accumulate in the plasma membrane and influence its structure and functions (fluidity, membrane proteins activities, signaling pathways), can have an impact on nerve impulses. It is currently thought that the ability of oxysterols to modulate nerve impulses could be explained by their influence on the characteristics and production of myelin as well as the functionality of Na+ and K+ channels

Profile of fatty acids, tocopherols, phytosterols and polyphenols in mediterranean oils (argan oils, olive oils, milk thistle seed oils and nigella seed oil) and evaluation of their antioxidant and cytoprotective activities

International audienceBackground: The effects of vegetable oils on human health depends on their components. Therefore, their profiles of lipid nutrients were determined. Objective: To establish and compare the fatty acid, tocopherol, phytosterol and polyphenol profiles of Mediterranean oils: cosmetic and dietary argan oils (AO; Morocco: Agadir, Berkane); olive oils (OO; Morocco, Spain, Tunisia); milk thistle seed oils (MTSO; Tunisia: Bizerte, Sousse, Zaghouane); nigella seed oil (NSO). Method: The biochemical profiles were determined by gas chromatography-flame ionization, high performance liquid chromatography and gas chromatography, coupled with mass spectrometry as required. The antioxidant and cytoprotective activities were evaluated with the KRL (Kit Radicaux Libres) and the fluorescein diacetate tests on nerve cells treated with 7-ketocholesterol (7KC). Results: The fatty acid profile revealed high linoleic acid (C18:2 n-6) content in AO, OO, MTSO and NSO. The highest levels of oleic acid (C18:1 n-9) were found in AO and OO. The tocopherol profile showed that Agadir AO contained the highest amount of α-tocopherol, also present at high level in MTSO and Tunisian OO; Berkane AO was rich in γ-tocopherol. The phytosterol profile indicated that β-sitosterol was predominant in the oils, except AO; spinasterol was only present in AO. Polyphenol profiles underlined that OO was the richest in polyphenols; hydroxytyrosol was only in OO; few polyphenols were detected in AO. The oils studied have antioxidant activities, and all of them, except NSO, prevented 7KC-induced cell death. The antioxidant characteristics of AO were positively correlated with procatechic acid and compestanol levels. Conclusion: Based on their biochemical profiles, antioxidant and cytoprotective characteristics, AO, OO, and MTSO are potentially beneficial to human health

Flavonoids differentially modulate liver X receptors activity—Structure-function relationship analysis

International audienceLiver X receptors (LXRs) α (NR1H3) and β (NR1H2) are nuclear receptors that have been involved in the regulation of many physiological processes, principally in the control of cholesterol homeostasis, as well as in the control of the cell death and proliferation balance. These receptors are thus promising therapeutic targets in various pathologies such as dyslipidemia, atherosclerosis, diabetes and/or cancers. These receptors are known to be activated by specific oxysterol compounds. The screening for LXR-specific ligands is a challenging process: indeed, these molecules should present a specificity towards each LXR-isoform. Because some natural products have significant effects in the regulation of the LXR-regulated homeostasis and are enriched in flavonoids, we have decided to test in cell culture the effects of 4 selected flavonoids (galangin, quercetin, apigenin and naringenin) on the modulation of LXR activity using double-hybrid experiments. In silico, molecular docking suggests specific binding pattern between agonistic and antagonistic molecules. Altogether, these results allow a better understanding of the ligand binding pocket of LXRα/β. They also improve our knowledge about flavonoid mechanism of action, allowing the selection and development of better LXR selective ligands