Corneliu N. Craciunescu

Dietary choline deficiency alters global and gene-specific DNA methylation in the developing hippocampus of mouse fetal brains

The availability of choline during critical periods of fetal development alters hippocampal development and affects memory function throughout life. Choline deficiency during fetal development reduces proliferation and migration of neuronal precursor cells in the mouse fetal hippocampus and these changes are associated with modifications in the protein levels of some cell cycle regulators and early differentiation markers. We fed C57 BL/6 mouse dams diets deficient or normal in choline content from days 12 to 17 of pregnancy, and then collected fetal brains on embryonic day 17. Using laser‐capture micro‐dissection we harvested cells from the ventricular and subventricular zones of Ammons horn and from the prime germinal zone of the dentate gyrus (hippocampus). In the ventricular and subventricular zones from the cholinedeficient group, we observed increased protein levels for kinase‐associated phosphatase (Kap) and for p15INK4b (two cell cycle inhibitors). In the dentate gyrus, we observed increased levels of calretinin (an early marker of neuronal differentiation). In fetal brain from mothers fed a choline‐deficient diet, DNA global methylation was decreased in the ventricular and sub‐ventricular zones of Ammons horn. We also observed decreased gene‐specific DNA methylation of the gene (Cdkn3) that encodes for Kap, correlating with increased expression of this protein. This was not the case for p15INK4b or calretinin (Cdkn2b and Calb2, respectively). These data suggest that choline deficiency‐induced changes in gene methylation could mediate the expression of a cell cycle regulator and thereby alter brain development. FASEB J. 20, 43–49 (2006)

Altered mitochondrial function and overgeneration of reactive oxygen species precede the induction of apoptosis by 1-O-octadecyl-2-methyl-rac-glycero-3-phosphocholine in p53-defective hepatocytes

The mechanism of induction of apoptosis by the novel anti‐cancer drug 1‐O‐octadecyl‐2‐methyl‐rac‐glycero‐3‐phosphocholine (ET‐18‐OCH3) was investigated in p53‐defective SV40 immortalized rat hepatocytes (CWSV1). Exposure to 12 μM ET‐18‐OCH3 for 36 h induced apoptosis as determined using classical morphological features and agarose gel electrophoresis of genomic DNA. Increased levels of reactive oxygen species (ROS) were detected spectrophotometrically using a nitroblue tetrazolium (NBT) assay in cells treated with ET‐18‐OCH3. Both the increased generation of ROS and the induction of apoptosis were inhibited when cells were treated concurrently with ET‐18‐OCH3 in the presence of the antioxidant α‐to‐copherol. Similar results were achieved when cells were switched acutely to choline‐deficient (CD) medium in the presence of the antioxidant. The possible role of mitochondria in the generation of ROS was investigated. Both ET‐18‐OCH3 and CD decreased the phos‐phatidylcholine (PC) content of mitochondrial and associated membranes, which correlated with depolarization of the mitochondrial membrane as analyzed using 5,5′,6,6′‐tetramethylbenzimidazolcarbocyanine iodide (JC‐1), a sensitive probe of mitochondrial membrane potential. Rotenone, an inhibitor of the mitochondrial electron transport chain, significantly reduced the in‐tracellular level of ROS and prevented mitochondrial membrane depolarization, correlating with a reduction of apoptosis in response to either ET‐18‐OCH3 or CD. Taken together, these results suggest that the form of p53‐independent apoptosis induced by ET‐18‐OCH3 is mediated by alterations in mitochondrial membrane PC, a loss of mitochondrial membrane potential, and the release of ROS, resulting in completion of apoptosis.— Vrablic, A. S., Albright, C. D., Craciunescu, C. N., Salganik, R. I., Zeisel, S. H. Altered mitochondrial function and overgeneration of reactive oxygen species precede the induction of apoptosis by 1‐O‐octadecyl‐2‐methyl‐rac‐glycero‐3‐phosphocholine in p53‐defective hepatocytes. FASEB J. 15, 1739—1744 (2001)

Choline deficiency alters global histone methylation and epigenetic marking at the Re1 site of the calbindin 1 gene

Maternal choline availability is essential for fetal neurogenesis. Choline deprivation (CD) causes hypomethylation of specific CpG islands in genes controlling cell cycling in fetal hippocampus. We now report that, in C57BL/6 mice, CD during gestational days 12–17 also altered methylation of the histone H3 in E17 fetal hippocampi. In the ventricular and subven‐tricular zones, monomethyl‐lysine 9 of H3 (H3K9me1) was decreased by 25% (P<0.01), and in the pyramidal layer, dimethyl‐lysine 9 of H3 (H3K9me2) was decreased by 37% (P<0.05). These changes were region specific and were not observed in whole‐brain preparations. Also, the same effects of CD on H3 methylation were observed in E14 neural progenitor cells (NPCs) in culture. Changes in G9a histone methyltransferase might mediate altered H3K9me2,1. Gene expression of G9a was decreased by 80% in CD NPCs (P<0.001). In CD, H3 was hypomethylated upstream of the RE1 binding site in the calbindin 1 promoter, and 1 CpG site within the calbindin1 promoter was hypermethylated. REST binding to RE1 (recruits G9a) was decreased by 45% (P<0.01) in CD. These changes resulted in increased expression of calbindin 1 in CD (260%; P< 0.05). Thus, CD modulates histone methylation in NPCs, and this could underlie the observed changes in neurogenesis.—Mehedint, M. G., Niculescu, M. D., Craciunescu, C. N., Zeisel, S. H. Choline deficiency alters global histone methylation and epigenetic marking at the Re1 site of the calbindin 1 gene. FASEB J. 24, 184–195 (2010). www.fasebj.org

Maternal dietary choline deficiency alters angiogenesis in fetal mouse hippocampus

We examined whether maternal dietary choline modulates angiogenesis in fetal brain. Pregnant C57BL/6 mice were fed either a choline-deficient (CD), control (CT), or choline-supplemented diet (CS) from days 12 to 17 (E12-17) of pregnancy and then fetal brains were studied. In CD fetal hippocampus, proliferation of endothelial cells (EC) was decreased by 32% (p < 0.01 vs. CT or CS) while differentiated EC clusters (expressing factor VIII related antigen (RA)) increased by 25% (p < 0.01 vs. CT or CS). These changes were associated with > 25% decrease in the number of blood vessels in CD fetal hippocampus (p < 0.01 vs. CT and CS), with no change in total cross-sectional area of these blood vessels. Expression of genes for the angiogenic signals derived from both endothelial and neuronal progenitor cells (NPC) was increased in CD fetal hippocampus VEGF C (Vegfc), 2.0-fold, p < 0.01 vs. CT and angiopoietin 2 (Angpt2), 2.1-fold, (p < 0.01 vs. CT)). Similar increased expression was observed in NPC isolated from E14 fetal mouse brains and exposed to low (5 μM), CT (70 μM), or high choline (280 μM) media for 72 h (low choline caused a 9.7-fold increase in relative gene expression of Vegfc (p < 0.001 vs. CT and high) and a 3.4-fold increase in expression of Angpt2, (p < 0.05 vs. CT and high). ANGPT2 protein was increased 42.2% (p < 0.01). Cytosine-phosphate-guanine dinucleotide islands in the proximity of the promoter areas of Vegfc and Angpt2 were hypomethylated in low choline NPC compared to CT NPC (p < 0.01). We conclude that maternal dietary choline intake alters angiogenesis in the developing fetal hippocampus.

Mitochondrial and microsomal derived reactive oxygen species mediate apoptosis induced by transforming growth factor-β1 in immortalized rat hepatocytes†

Transforming growth factor‐β1 (TGFβ1) is a multifunctional cytokine that is over expressed during liver hepatocytes injury and regeneration. SV40‐transformed CWSV‐1 rat hepatocytes that are p53‐defective undergo apoptosis in response to choline deficiency (CD) or TGFβ1, which mediates CD‐apoptosis. Reactive oxygen species (ROS) are essential mediators of apoptosis. We have shown that apoptosis induced by TGFβ1 is accompanied by ROS generation and the ROS‐trapping agent N‐acetylcysteine (NAC) inhibits TGFβ1‐induced apoptosis. While persistent induction of ROS contributes to this form of apoptosis, the source of ROS generated downstream of TGFβ1 is not clear. The mitochondria and the endoplasmic reticulum both harbor potent electron transfer chains that might be the source of ROS essential for completion of TGFβ1‐apoptosis. Here we show that CWSV‐1 cells treated with cyclosporine A, which prevents opening of mitochondrial membrane pores required for ROS generation, inhibits TGFβ1‐induced apoptosis. A similar effect was obtained by treating these cells with rotenone, an inhibitor of complex 1 of the mitochondrial electron transfer chain. However, we demonstrate that TGFβ1 induces cytochrome P450 1A1 and that metyrapone, a potent inhibitor of cytochrome P450 1A1, inhibits TGFβ1‐induced apoptosis. Therefore, our studies indicate that concurrent with promoting generation of ROS from mitochondria, TGFβ1 also promotes generation of ROS from the cytochrome P450 electron transfer chain. Since inhibition of either of these two sources of ROS interferes with apoptosis, it is reasonable to conclude that the combined involvement of both pathways is essential for completion of TGFβ1‐induced apoptosis. J. Cell. Biochem. 89: 254–261, 2003.

Perinatal manipulation of α-linolenic acid intake induces epigenetic changes in maternal and offspring livers

Previous studies indicated that the intake of α‐linolenic acid (ALA) can alter the concentration of both ω‐6 and ω‐3 fatty acids in both mother and offspring, with consequences on postnatal brain development. This study describes the association between maternal ALA availability during gestation and lactation, and alterations in the Fads2 DNA methylation in both maternal and offspring livers, at the end of lactation period. Both Fads2 promoter and intron 1 DNA methylation were increased in the groups receiving postnatal flaxseed oil containing 50% ALA (mothers or pups), while bivariate analysis indicated a significant association of the Fads2 epigenetic status in the liver between each mother and its offspring. In addition, Fads2 expression was negatively correlated with promoter methylation at the individual level in maternal livers (P<0.05). This study also indicated that the interplay between ALA availability during gestation and lactation can differentially alter the expression of desaturases and elongases involved in ω‐6 and ω‐3 metabolic pathways. In summary, when considering the perinatal dietary ALA requirements in mice, both gestation and lactation periods should be considered as having distinct roles in modulating the metabolism of ω‐6 and ω‐3 fatty acids in maternal mouse livers.—Niculescu, M. D., Lupu, D. S., Craciunescu, C. N. Perinatal manipulation of α‐linolenic acid intake induces epigenetic changes in maternal and offspring livers. FASEB J. 27, 350–358 (2013). www.fasebj.org

Deletion of murine choline dehydrogenase results in diminished sperm motility

Choline dehydrogenase (CHDH) catalyzes the conversion of choline to betaine, an important methyl donor and organic osmolyte. We have previously identified single nucleotide polymorphisms (SNPs) in the human CHDH gene that, when present, seem to alter the activity of the CHDH enzyme. These SNPs occur frequently in humans. We created a Chdh(-/-) mouse to determine the functional effects of mutations that result in decreased CHDH activity. Chdh deletion did not affect fetal viability or alter growth or survival of these mice. Only one of eleven Chdh(-/-) males was able to reproduce. Loss of CHDH activity resulted in decreased testicular betaine and increased choline and PCho concentrations. Chdh(+/+) and Chdh(-/-) mice produced comparable amounts of sperm; the impaired fertility was due to diminished sperm motility in the Chdh(-/-) males. Transmission electron microscopy revealed abnormal mitochondrial morphology in Chdh(-/-) sperm. ATP content, total mitochondrial dehydrogenase activity and inner mitochondrial membrane polarization were all significantly reduced in sperm from Chdh(-/-) animals. Mitochondrial changes were also detected in liver, kidney, heart, and testis tissues. We suggest that men who have SNPs in CHDH that decrease the activity of the CHDH enzyme could have decreased sperm motility and fertility.

Regulation of choline deficiency apoptosis by epidermal growth factor in CWSV-1 rat hepatocytes.

Previous studies show that acute choline deficiency (CD) triggers apoptosis in cultured rat hepatocytes (CWSV-1 cells). We demonstrate that prolonged EGF stimulation (10 ng/mL x 48 hrs) restores cell proliferation, as assessed by BrdU labeling, and protects cells from CD-induced apoptosis, as assessed by TUNEL labeling and cleavage of poly(ADP-ribose) polymerase. However, EGF rescue was not accompanied by restoration of depleted intracellular concentrations of choline, glycerphosphocholine, phosphocholine, or phosphatidylcholine. In contrast, we show that EGF stimulation blocks apoptosis by restoring mitochondrial membrane potential (Δ Ψm), as determined using the potential-sensitive dye chloromethyl-X-rosamine, and by preventing the release and nuclear localization of cytochrome c. We investigated whether EGF rescue involves EGF receptor phosphorylation and activation of the down-stream cell survival factor Akt. Compared to cells in control medium (CT, 70 μmol choline x 48hrs), cells in CD medium (5 μmol choline) were less sensitive to EGF-induced (0-300 ng/mL x 5 min) receptor tyrosine phosphorylation. Compared to cells in CT medium, cells in CD medium treated with EGF (10 ng/mL x 5 min) exhibited higher levels of phosphatidylinositol 3-kinase (PI3K)-dependent phosphorylation of AktSer473. Inactivation of PI3K was sufficient to block EGF-stimulated activation of Akt, restoration of mitochondrial Δ Ψm, and prevention of cytochrome c release. These studies indicate that stimulation with EGF activates a cell survival response against CD-apoptosis by restoring mitochondrial membrane potential and preventing cytochrome c release and nuclear translocation which are mediated by activation of Akt in hepatocytes.

Choline availability during embryonic development alters the localization of calretinin in developing and aging mouse hippocampus

Abstract Choline availability in the diet during pregnancy alters fetal brain biochemistry with resulting behavioral changes that persist throughout the lifetime of the offspring. In the present study, the effects of dietary choline on the onset of GABAergic neuronal differentiation in developing fetal brain, as demarcated by the expression of calcium binding protein calretinin, are described. In these studies, timed-pregnant mice were fed choline supplemented, control or choline deficient AIN-76 diet from day 12-17 of pregnancy and the brains of their fetuses were studied on day 17 of gestation. In the primordial dentate gyrus, we found that pups from choline deficient-dams had more calretinin protein (330% increase), and pups from choline supplemented-dams had less calretinin protein (70% decrease), than did pups from control-dams. Importantly, decreased calretinin protein was still detectable in hippocampus in aged, 24-month-old mice, born of choline supplemented-dams and maintained since birth on a control diet. Thus, alterations in the level of calretinin protein in fetal brain hippocampus could underlie the known, life long effects of maternal dietary choline availability on brain development and behavior.

Dietary Docosahexaenoic Acid Supplementation Modulates Hippocampal Development in the Pemt−/− Mouse

The development of fetal brain is influenced by nutrients such as docosahexaenoic acid (DHA, 22:6) and choline. Phosphatidylethanolamine-N-methyltransferase (PEMT) catalyzes the biosynthesis of phosphatidylcholine from phosphatidylethanolamine enriched in DHA and many humans have functional genetic polymorphisms in the PEMT gene. Previously, it was reported that Pemt−/− mice have altered hippocampal development. The present study explores whether abnormal phosphatidylcholine biosynthesis causes altered incorporation of DHA into membranes, thereby influencing brain development, and determines whether supplemental dietary DHA can reverse some of these changes. Pregnant C57BL/6 wild type (WT) and Pemt−/− mice were fed a control diet, or a diet supplemented with 3 g/kg of DHA, from gestational day 11 to 17. Brains from embryonic day 17 fetuses derived from Pemt−/− dams fed the control diet had 25–50% less phospholipid-DHA as compared with WT (p < 0.05). Also, they had 60% more neural progenitor cell proliferation (p < 0.05), 60% more neuronal apoptosis (p < 0.01), and 30% less calretinin expression (p < 0.05; a marker of neuronal differentiation) in the hippocampus compared with WT. The DHA-supplemented diet increased fetal brain Pemt−/− phospholipid-DHA to WT levels, and abrogated the neural progenitor cell proliferation and apoptosis differences. Although this diet did not change proliferation in the WT group, it halved the rate of apoptosis (p < 0.05). In both genotypes, the DHA-supplemented diet increased calretinin expression 2-fold (p < 0.05). These results suggest that the changes in hippocampal development in the Pemt−/− mouse could be mediated by altered DHA incorporation into membrane phospholipids, and that maternal dietary DHA can influence fetal brain development.