Cross-Talk between miRNAs from the Dlk1-Dio3 Locus and Histone Methylation to Protect Male Cerebellum from Methyl Donor Deficiency.

SCOPE: Disruption of the one carbon metabolism during development, i.e., following a gestational vitamin B9 and B12 deficiencies, is involved in birth defects and brain development delay. Using a rat nutritional model, consisting of pups born to dams fed a vitamin B9 and B12 deficient diet (MDD), the study previously reports molecular and cellular alterations in the brain, in a sex dependent manner, with females being more affected than males. The study hypothesizes that epigenetic modifications could participate in the sex differences is observed. METHODS AND RESULTS: The study investigates lysine methylation of histones and expression of microRNAs in the cerebellum of MDD male and female pups. The study reports a differential regulation of H3K36Me2 and H4K20Me3 between males and females, in response to MDD. Moreover, distinct regulation of Kmt5b and Kdm2a expression by miR-134-5p and miR-369-5p from the Dlk1-Dio3 locus, contributes to the maintenance of expression of genes involved in synaptic plasticity. CONCLUSION: These results could explain the neuroprotection to MDD that male pups display. The work will contribute to the understanding of the consequences of vitamin starvation on brain development, as well as how the epigenome is affected by one carbon metabolism disruption.

Consequences of Paternal Nutrition on Offspring Health and Disease.

It is well established that the maternal diet during the periconceptional period affects the progeny's health. A growing body of evidence suggests that the paternal diet also influences disease onset in offspring. For many years, sperm was considered only to contribute half of the progeny's genome. It now appears that it also plays a crucial role in health and disease in offspring's adult life. The nutritional status and environmental exposure of fathers during their childhood and/or the periconceptional period have significant transgenerational consequences. This review aims to describe the effects of various human and rodent paternal feeding patterns on progeny's metabolism and health, including fasting or intermittent fasting, low-protein and folic acid deficient food, and overnutrition in high-fat and high-sugar diets. The impact on pregnancy outcome, metabolic pathways, and chronic disease onset will be described. The biological and epigenetic mechanisms underlying the transmission from fathers to their progeny will be discussed. All these data provide evidence of the impact of paternal nutrition on progeny health which could lead to preventive diet recommendations for future fathers.

Wnt Signaling Pathways Are Dysregulated in Rat Female Cerebellum Following Early Methyl Donor Deficiency.

Gestational methyl donor (especially B9 and B12 vitamins) deficiency is involved in birth defects and brain development retardation. The underlying molecular mechanisms that are dysregulated still remain poorly understood, in particular in the cerebellum. As evidenced from previous data, females are more affected than males. In this study, we therefore took advantage of a validated rat nutritional model and performed a microarray analysis on female progeny cerebellum, in order to identify which genes and molecular pathways were disrupted in response to methyl donor deficiency. We found that cerebellum development is altered in female pups, with a decrease of the granular cell layer thickness at postnatal day 21. Furthermore, we investigated the involvement of the Wnt signaling pathway, a major molecular pathway involved in neuronal development and later on in synaptic assembly and neurotransmission processes. We found that Wnt canonical pathway was disrupted following early methyl donor deficiency and that neuronal targets were selectively enriched in the downregulated genes. These results could explain the structural brain defects previously observed and highlighted new genes and a new molecular pathway affected by nutritional methyl donor deprivation.

Early methyl donor deficiency alters cAMP signaling pathway and neurosteroidogenesis in the cerebellum of female rat pups.

Early deficiency of the methyl donors folate and vitamin B12 produces hyperhomocysteinemia and cognitive and motor disorders in 21-day-old rat pups from dams fed a diet deficient in methyl donors during gestation and lactation. These disorders are associated with impaired neurogenesis and altered synaptic plasticity in cerebellum. We aimed to investigate whether these disorders could be related to impaired expression of neurosteroidogenesis-associated proteins, key regulator receptors, and some steroid content in the cerebellum. The methyl donor deficiency produced a decreased concentration of folate and vitamin B12, along with accumulation of homocysteine in Purkinje cells in both sexes, whereas the S-adenosylmethionine/S-adenosylhomocysteine ratio was reduced only in females. The transcription level and protein expression of StAR, aromatase, ERα, ERß, and LH receptors were decreased only in females, with a marked effect in Purkinje cells, as shown by immunohistochemistry. Consistently, reduced levels of estradiol and pregnenolone were measured in cerebellar extracts of females only. The decreased expression levels of the transcriptional factors CREB, phospho-CREB, and SF-1, the lesser increase of cAMP concentration, and the lower level of phospho-PKC in the cerebellum of deficient females suggest that the activation of neurosteroidogenesis via cAMP-mediated signaling pathways associated with LHR activation would be altered. In conclusion, a gestational methyl donor deficiency impairs neurosteroidogenesis in cerebellum in a sex-dependent manner.

Gestational methyl donor deficiency alters key proteins involved in neurosteroidogenesis in the olfactory bulbs of newborn female rats and is associated with impaired olfactory performance.

Gestational methyl donor deficiency (MDD) leads to growth retardation as well as to cognitive and motor disorders in 21-d-old rat pups. These disorders are related to impaired neurogenesis in the cerebral neurogenic areas. Olfactory bulbs (OB), the main target of neuronal progenitors originating from the subventricular zone, play a critical role during the postnatal period by allowing the pups to identify maternal odour. We hypothesised that growth retardation could result from impaired suckling due to impaired olfactory discrimination through imbalanced apoptosis/neurogenesis in the OB. Since neurosteroidogenesis modulates neurogenesis in OB, in the present study, we investigated whether altered neurosteroidogenesis could explain some these effects. Pups born to dams fed a normal diet (n 24) and a MDD diet (n 27) were subjected to olfactory tests during the lactation and weaning periods (n 24 and 20, respectively). We studied the markers of apoptosis/neurogenesis and the expression levels of the key neurosteroidogenic enzyme aromatase, the cholesterol-transfer protein StAR (steroidogenic acute regulatory protein) and the ERα oestrogen receptor and the content of oestradiol in OB. The 21-d-old MDD female pups displayed lower body weight and impaired olfactory discrimination when compared with the control pups. MDD led to greater homocysteine accumulation and more pronounced apoptosis, along with impaired cell proliferation in the OB of female pups. The expression levels of aromatase, StAR and ERα as well as the content of oestradiol were lower in the OB of the MDD female pups than in those of the control female pups. In conclusion, gestational MDD may alter olfactory discrimination performances by affecting neurogenesis, apoptosis and neurosteroidogenesis in OB in a sex-dependent manner. It may be involved in growth retardation through impaired suckling.

N-truncated amyloid-beta oligomers induce learning impairment and neuronal apoptosis.

N-terminal-truncated forms of amyloid-beta (A beta) peptide have been recently suggested to play a pivotal role early in Alzheimer's disease (AD). Among them, A beta 3(pE)-42 peptide, starting with pyroglutamyl at residue Glu-3, is considered as the predominant A beta species in AD plaques and pre-amyloid lesions. Its abundance is reported to be directly proportional to the severity of the clinical phenotype. The present study investigates the effects of soluble oligomeric A beta 3(pE)-42 after intracerebroventricular injection on mice learning ability and the molecular mechanisms of its in vitro neurotoxicity. Mice injected with soluble A beta 3(pE)-42 or A beta(l-42) displayed impaired spatial working memory and delayed memory acquisition in Y-maze and Morris water maze tests, while those injected with soluble A beta(42-1) showed no effect. These cognitive alterations were associated with free radical overproduction in the hippocampus and olfactory bulbs, but not in the cerebral cortex or cerebellum. In vitro, A beta 3(pE)-42 oligomers induced a redox-sensitive neuronal apoptosis involving caspase activation and an arachidonic acid-dependent pro-inflammatory pathway. These data suggest that A beta 3(pE)-42 could mediate the neurodegenerative process and subsequent cognitive alteration occurring in preclinical AD stages.

Soluble oligomers of amyloid-beta peptide induce neuronal apoptosis by activating a cPLA2-dependent sphingomyelinase-ceramide pathway.

Recent data have revealed that soluble oligomeric amyloid-beta peptide (Abeta) may be the proximate effectors of neuronal injuries and death in Alzheimer's disease (AD) by unknown mechanisms. Consistently, we recently demonstrated the critical role of a redox-sensitive cytosolic calcium-dependent phospholipase A2 (cPLA2)-arachidonic acid (AA) pathway in Abeta oligomer-induced cell death. According to the involvement of oxidative stress and polyunsaturated fatty acids like AA in the regulation of sphingomyelinase (SMase) activity, the present study underlines the role of SMases in soluble Abeta-induced apoptosis. Soluble Abeta oligomers induced the activation of both neutral and acidic SMases, as demonstrated by the direct measurement of their enzymatic activities, by the inhibitory effects of both specific neutral and acidic SMase inhibitors, and by gene knockdown using antisense oligonucleotides. Furthermore, soluble Abeta-mediated activation of SMases and subsequent cell death were found to be inhibited by antioxidant molecules and a cPLA2-specific inhibitor or antisense oligonucleotide. We also demonstrate that sphingosine-1-phosphate is a potent neuroprotective factor against soluble Abeta oligomer-induced cell death and apoptosis by inhibiting soluble Abeta-induced activation of acidic sphingomyelinase. These results suggest that Abeta oligomers induce neuronal death by activating neutral and acidic SMases in a redox-sensitive cPLA2-AA pathway.

Docosahexaenoic acid prevents neuronal apoptosis induced by soluble amyloid-beta oligomers.

A growing body of evidence supports the notion that soluble oligomers of amyloid-beta (Abeta) peptide interact with the neuronal plasma membrane, leading to cell injury and inducing death-signalling pathways that could account for the increased neurodegeneration occurring in Alzheimer's disease (AD). Docosahexaenoic acid (DHA, C22:6, n-3) is an essential polyunsaturated fatty acid in the CNS and has been shown in several epidemiological and in vivo studies to have protective effects against AD and cognitive alterations. However, the molecular mechanisms involved remain unknown. We hypothesized that DHA enrichment of plasma membranes could protect neurones from apoptosis induced by soluble Abeta oligomers. DHA pre-treatment was observed to significantly increase neuronal survival upon Abeta treatment by preventing cytoskeleton perturbations, caspase activation and apoptosis, as well as by promoting extracellular signal-related kinase (ERK)-related survival pathways. These data suggest that DHA enrichment probably induces changes in neuronal membrane properties with functional outcomes, thereby increasing protection from soluble Abeta oligomers. Such neuroprotective effects could be of major interest in the prevention of AD and other neurodegenerative diseases.

Synthesis and in vitro antioxidant activity of glycyrrhetinic acid derivatives tested with the cytochrome P450/NADPH system.

Five glycyrrhetinic acid (Ib) derivatives have been synthesized to try to improve the antioxidant activity. Their in vitro antioxidant activities were studied using a cytochrome P450/NADPH reductase system from rat liver microsomes. The generation of microsomal free radicals was followed by oxidation of the DCFH-DA probe, while evaluating the capacity to inhibit reactive oxygen species (ROS) formation. Two hydroxylated derivatives, 18beta-olean-12-ene-3beta,11alpha,30-triol (II) and 18beta-olean-12-ene-3beta,11beta,30-triol (IV), exhibited strong antioxidant activities. At a concentration of 1.0 mg/ml, these derivatives inhibited ROS formation by 50% and 51%, respectively. Moreover, two homo- and heterocyclic diene derivatives, 18beta-olean-11,13(18)-diene-3beta,30-diol (III) and 18beta-olean-9(11),12-diene-3beta,30-diol (V), were also effective in ROS-scavenging activity (inhibition of 41% and 44% of ROS activity, respectively). In the same conditions, the lead compound (Ib) and the reference vitamin E inhibited ROS activity by 31% and 32%, respectively. Our results suggest that the chemical reduction of the 11-keto and 30-carboxyl groups into hydroxyl function (example, II, IV) can increase the antioxidant activity of Ib significantly. In view of these results, our study represents a further approach to the development of potential therapeutic agents from Ib derivatives for use in pathologic events in which, free radical damage could be involved.

Cytochrome p-450-mediated differential oxidative modification of proteins: albumin, apolipoprotein E, and CYP2E1 as targets.

Although many studies established a role of cytochrome P-450s in metabolism of xenobiotics, few studies evaluating the ability of cytochrome P-450s to oxidize proteins have been reported. The ability of cytochrome P-450s to induce oxidative modification of albumin, apolipoprotein E, and CYP2E1 protein was investigated. Microsomal cytochrome P-450s induced production of reactive radical species, leading to differential modification of the proteins. Albumin remained unmodified, and CYP2E1 protein was degraded, whereas recombinant and endogenous apolipoprotein E was aggregated. The modification of apolipoprotein E was isoform independent. Cytochrome P-450 inhibitors or antioxidants inhibited the production of reactive radical species and protein modification. These results demonstrate that response of each protein to cytochrome P-450-mediated oxidative attack is different, and cytochrome P-450s can induce apolipoprotein E aggregation, a process that might be relevant to accumulation of altered protein in various abnormal conditions. In view of the ubiquitous expression of cytochrome P-450s, the present results may have important toxicological implications.

Myeloperoxidase G-463A polymorphism and Alzheimer's disease in the ApoEurope study.

Alzheimer's disease (AD) is the most common cause of dementia in the elderly. Epidemiological and molecular genetic studies have shown the existence of several genes associated with increased risk of AD, the major genetic susceptibility locus coding for apolipoprotein E (apoE). A polymorphism in the myeloperoxidase gene (MPO) has previously been associated with AD susceptibility. However, results in the literature are controversial and seem to be dependent on several factors such as gender, apoE polymorphism or the genetic structure of the population. We investigated MPO G-463A and apoE polymorphism in 265 cases and 246 controls from the ApoEurope Study. In females, we found a significant association between MPO genotype and AD (P=0.034), GG genotype frequency being lower in cases (52.4%) as compared to controls (64.2%). In men, there was no significant effect of MPO polymorphism. No interaction was found between MPO polymorphism and apoE epsilon 4 allele. In conclusion, the G-463A polymorphism of MPO was statistically associated with AD in a gender-specific manner. However, given the low significance of P value we suggest no causal effect of the MPO gene in AD, as also evidenced in a recent meta-analysis. Our results support the hypothesis of a possible linkage disequilibrium between the MPO G-463A gene polymorphism and another functional variant involved in AD.

Myeloperoxidase polymorphisms in brain infarction. Association with infarct size and functional outcome.

Myeloperoxidase (MPO) has been shown to contribute to several diseases and more particularly to atherosclerosis through excessive ROS production via the MPO/H(2)O(2)/Cl(-) oxidation system. The aim of this study was to determine whether there is an association between MPO polymorphisms and brain infarction (BI), one of the main consequences of atherosclerosis. We investigated MPO G-463A and G-129A polymorphisms in 450 patients with BI confirmed by magnetic resonance imaging (MRI) and 450 controls of the GENIC (Génétique de l'Infarctus Cérébral) Study. Genotype determination of MPO was performed by polymerase chain reaction and allele-specific oligonucleotide hybridization (ASO). Genotype distributions for each of both MPO polymorphisms were found to be similar between cases and controls overall, and according to etiologic subtypes or gender. The frequency of the A allele of the G-463A polymorphism was 22% (95% confidence interval, 19.4 to 24.9) and the frequency of the A allele of the G-129A polymorphism was 6.8% (95% confidence interval, 5.3 to 8.6). The odds ratio (OR) for BI in carriers of the A allele of the G-129A polymorphism was 0.92 (95% confidence interval, 0.61 to 1.39), and the OR for BI in carriers of the A allele of the G-463A polymorphism was 1.15 (95% confidence interval, 0.88 to 1.52). No association between the main risk factors for BI such as hypertension, cholesterol, diabetes and MPO polymorphisms was found. In analyses restricted to cases, we identified an association between the A allele of the G-129A polymorphism and the size of the brain infarct (P=0.01). Furthermore, the A allele of the G-463A polymorphism was associated with a poorer functional short-term outcome as evaluated by the Rankin score (P=0.02). In conclusion, MPO polymorphisms were associated with the extent of brain damage and the functional outcome rather than with the risk of developing a BI.

Differential role of CYP2E1 binders and isoniazid on CYP2E1 protein modification in NADPH-dependent microsomal oxidative reactions: free radical scavenging ability of isoniazid.

We evaluated the effect of "weak" CYP2E1 binders (ethanol, acetone and glycerol) "tight" CYP2E1 binders (4-methylpyrazole, imidazole, isoniazid and pyridine) and CCl4 (suicide substrate of CYP2E1) on the NADPH-dependent production of microsomal reactive oxygen species (ROS), lipid peroxidation (LPO), and subsequent modification of microsomal and CYP2E1 proteins. The oxidation of 2',7'-dichlorofluorescin diacetate (DCFHDA) was used as an index of formation of microsomal ROS and LPO-derived reactive species. Microsomal LPO was determined by malondialdehyde (MDA) HPLC measurement. Addition of NADPH to rat liver microsomes initiated DCFHDA oxidation and MDA formation, leading to further selective modification of microsomal proteins and proteases-independent degradation of CYP2E1 protein. Iron chelators prevented these processes whereas hydroxyl radical scavengers showed weak effects, suggesting an important role of LPO. Among the tested CYP2E1 binders, only isoniazid strongly inhibited NADPH-dependent DCFHDA oxidation, LPO and modification of microsomal proteins. Other CYP2E1 binders showed weak inhibitory effects of these processes. Concerning NADPH-dependent modification of CYP2E1 protein, all of the tested CYP2E1 binders, except glycerol, prevented this process with a different potency (isoniazid > 4-methylpyrazole = imidazole = pyridine 3 >> acetone > ethanol). "Tight" binders were more effective than "weak" binders. The CCl4 stimulated the DCFHDA oxidation, LPO and CYP2E1 protein modification. Among the tested CYP2E1 binders, only isoniazid effectively scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals. In microsomes isolated from CYP2E1 transfected HepG2 cells, isoniazid inhibited the CYP2E1-dependent DCFHDA oxidation whereas other CYP2E1 binders did not inhibit this reaction although these compounds strongly inhibited CYP2E1 activity. The present study demonstrates that CYP2E1 binders and isoniazid differentially inhibit LPO-catalyzed oxidative modification of CYP2E1 protein in NADPH-dependent microsomal reactions. It seems that CYP2E1 binders protect CYP2E1 from the oxidative modification mainly by binding to the active site of the enzyme, rather than by blocking the reactive species production. The strong protective effect of isoniazid can be attributed to its ability to scavenge free radicals. These effects of CYP2E1 binders are considered to contribute to the regulation of hepatic CYP2E1 protein levels via stabilization of the protein.

Growing significance of myeloperoxidase in non-infectious diseases.

Myeloperoxidase (MPO) is a glycoprotein released by activated polymorphonuclear neutrophils, which takes part in the defense of the organism through production of hypochlorous acid (HOCl), a potent oxidant. Since the discovery of MPO deficiency, initially regarded as rare and restricted to patients suffering from severe infections, MPO has attracted clinical attention. The development of new technologies allowing screening for this defect has permitted new advances in the comprehension of underlying mechanisms. Apart from its implications for host defense, the expression of MPO restricted to myeloid precursors makes MPO mRNA a good marker of acute myeloid leukemia. In addition, during the last few years, involvement of MPO has been described in numerous diseases such as atherosclerosis, lung cancer, Alzheimer's disease and multiple sclerosis. Both strong oxidative activity and MPO genetic polymorphism have been involved. This review summarizes the broad range of diseases involving MPO and points out the possible use of this protein as a new clinical marker and a future therapeutic target.