Craig D. Albright

Choline availability alters embryonic development of the hippocampus and septum in the rat

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 cell proliferation, migration, and apoptosis in neuronal progenitor cells in the hippocampus and septum were analyzed in fetal brains at different stages of embryonic development. Timed-pregnant rats on day E12 were fed AIN-76 diet with varying levels of dietary choline for 6 days, and, on days E18 or E20, fetal brain sections were collected. We found that choline deficiency (CD) significantly decreased the rate of mitosis in the neuroepithelium adjacent to the hippocampus. An increased number of apoptotic cells were found in the region of the dentate gyrus of CD hippocampus compared to controls (5.5+/-0.7 vs. 1.9+/-0.3 apoptotic cells per section; p<0.01). Using a combination of bromodeoxyuridine (BrdU) labeling and an unbiased computer-assisted image analysis method, we found that modulation of dietary choline availability changed the distribution and migration of precursor cells born on E16 in the fimbria, primordial dentate gyrus, and Ammons horn of the fetal hippocampus. CD also decreased the migration of newly born cells from the neuroepithelium into the lateral septum, thus indicating that the sensitivity of fetal brain to choline availability is not restricted to the hippocampus. We found an increase in the expression of TOAD-64 protein, an early neuronal differentiation marker, in the hippocampus of CD day E18 fetal brains compared to controls. These results show that dietary choline availability alters the timing of the genesis, migration, and commitment to differentiation of progenitor neuronal-type cells in fetal brain hippocampal regions known to be associated with learning and memory processes in adult brain.

Maternal dietary choline availability alters mitosis, apoptosis and the localization of TOAD-64 protein in the developing fetal rat septum

Maternal changes in dietary choline availability alter brain biochemistry and hippocampal development in the offspring resulting in lifelong behavioral changes in the offspring. In order to better understand the relationship between maternal diet, brain cytoarchitecture and behavior, we investigated the effects of choline availability on cell proliferation, apoptosis and differentiation in the fetal rat brain septum. Timed-pregnant rats on day E12 were fed AIN-76 diet with varying levels of dietary choline for 6 days. We found that choline deficiency (CD) significantly decreased the rate of mitosis in the progenitor neuroepithelium adjacent to the septum. In addition, we found an increased number of apoptotic cells in the septum of CD animals compared to controls (3.5+/-0.5 vs. 1.7+/-0.5 apoptotic cells per section; p<0.05). However, CD had no effect on apoptosis in the indusium griseum (IG), a region of cortex dorsal to the septum. Using an unbiased image analysis method and a monoclonal antibody we found a decreased expression of the TOAD-64 kDa protein, a marker of commitment to neuronal differentiation during fetal development, in the dorsal lateral septum of CD animals. CD also decreased the expression of TOAD-64 kDa protein in the IG and cortical plate adjacent to the septum. These results show that dietary choline availability during pregnancy alters the timing of mitosis, apoptosis and the early commitment to neuronal differentiation by progenitor cells in regions of the fetal brain septum, as well as hippocampus, two brain regions known to be associated with learning and memory.

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 induces apoptosis in SV40-immortalized CWSV-1 rat hepatocytes in culture.

Immortalized CWSV‐1 rat hepatocytes, in which p53 protein is inactivated by SV40 large T antigen, had increased numbers of cells with strand breaks in genomic DNA (terminal dUTP end labeling) when grown in 0 μM choline (67‐73% of cells) than when grown in 70 μM choline (2‐3% of cells). Internucleosomal fragmentation of DNA (DNA ladders) was detected in cells grown with 5 μM and 0 μM choline for 72 h. Cells treated with 0 or 5 μM choline for 72 h detached from the substrate in high numbers (58% of choline deficient cells vs. 1.4% of choline sufficient cells detached) exhibited a high incidence of apoptosis (apoptotic bodies were seen in 55‐75% of cells; 67‐73% had DNA strand breaks), and an absence of mitosis and proliferating cell nuclear antigen (PCNA) expression. Cells undergoing DNA fragmentation had functioning mitochondria. At 24 h, cells grown in 0 or 5 μM choline synthesized DNA more rapidly than those grown in 70 μM choline. By 72 h, the cells grown in 0 or 5 μM choline were forming DNA much more slowly than control cells (assessed by thymidine incorporation, PCNA expression, and mitotic index). Western blot analysis showed that p53 in the nucleus of cells was detected in direct association with SV40 T‐antigen, and was therefore likely to be inactive. We conclude that choline deficiency kills CWSV‐1 hepatocytes in culture by inducing apoptosis via what may be a p53‐independent process, and that this process begins in viable cells before they detach from the culture dish.—Albright, C. D., Liu, R., Bethea, T. C., da Costa, K.‐A., Salganik, R. I., Zeisel, S. H. Choline deficiency induces apoptosis in SV40‐im‐ mortalized CWSV‐1 rat hepatocytes in culture. FASEBJ. 10,510‐516(1996)

Apoptosis is induced by choline deficiency in fetal brain and in PC12 cells

Treatment of rats with choline during critical periods in brain development results in long-lasting enhancement of spatial memory in their offspring. Apoptosis is a normal process during brain development, and, in some tissues, is modulated by the availability of the nutrient choline. In these studies, we examined whether availability of choline influences apoptosis in fetal brain and in the PC12 cell line derived from a rat pheochromocytoma. Timed-bred Sprague Dawley rats were fed a choline-deficient (CD), choline-control, or choline-supplemented (CS) diet for 6 days and, on embryonic day 18, fetal brain slices were prepared and apoptosis was assessed using terminal dUTP nucleotide end labeling (TUNEL) to detect DNA strand breaks and by counting of apoptotic bodies. TUNEL-positive cells were detected in 15.9% (P < 0.01), 8.7% and 7.2% of hippocampal cells from fetuses of dams fed the CD, control or CS diets, respectively. A similar inverse relationship between dietary intake of choline and TUNEL positive cells was detected in an area of cerebral cortex from these fetal brain slices. Counts of apoptotic bodies in fetal brain slices correlated inversely with choline intake of the mothers (6.2% (P < 0.01), 2.5% and 1.9% of hippocampal cells had apoptotic bodies in fetuses of dams fed the CD, control and CS diets, respectively). PC12 cells were grown in DMEM/F12 media supplemented with 70 microM choline or with 0 microM choline. The number of apoptotic bodies in PC12 cells increased when cells were grown in 0 microM choline medium (1.5%; P < 0.05) compared to 70 microM choline medium (0.55%). In PC12 cells, TUNEL labeling (DNA strand breaks) increased in choline deficient (13.5%, P < 0.05) compared to sufficient medium (5.0%). In addition, cleavage of genomic DNA-into 200 bp internucleosomal fragments was detected in choline-deficient cells. These results show that choline deficiency induces-apoptotic cell death in neuronal-type cells and in whole brain. We suggest that variations in choline availability to brain modulate apoptosis rates during development.

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.

Methyl-group donors cannot prevent apoptotic death of rat hepatocytes induced by choline-deficiency

Choline‐deficiency causes liver cells to die by apoptosis, and it has not been clear whether the effects of choline‐deficiency are mediated by methyl‐deficiency or by lack of choline moieties. SV40 immortalized CWSV‐1 hepatocytes were cultivated in media that were choline‐sufficient, choline‐deficient, choline‐deficient with methyl‐donors (betaine or methionine), or choline‐deficient with extra folate/vitamin B12. Choline‐deficient CWSV‐1 hepatocytes were not methyl‐deficient as they had increased intracellular S‐adenosylmethionine concentrations (132% of control; P < 0.01). Despite increased phosphatidylcholine synthesis via sequential methylation of phosphatidylethanolamine, choline‐deficient hepatocytes had significantly decreased (P < 0.01) intracellular concentrations of choline (20% of control), phosphocholine (6% of control), glycerophosphocholine (15% of control), and phosphatidylcholine (55% of control). Methyl‐supplementation in choline‐deficiency enhanced intracellular methyl‐group availability, but did not correct choline‐deficiency induced abnormalities in either choline metabolite or phospholipid content in hepatocytes. Methyl‐supplemented, choline‐deficient cells died by apoptosis. In a rat study, 2 weeks of a choline‐deficient diet supplemented with betaine did not prevent the occurrence of fatty liver and the increased DNA strand breakage induced by choline‐deficiency. Though dietary supplementation with betaine restored hepatic betaine concentration and increased hepatic S‐adenosylmethionine/S‐adenosylhomocysteine ratio, it did not correct depleted choline (15% of control), phosphocholine (6% control), or phosphatidylcholine (48% of control) concentrations in deficient livers. These data show that decreased intracellular choline and/or choline metabolite concentrations, and not methyl deficiency, are associated with apoptotic death of hepatocytes. J. Cell. Biochem, 64:196–208.

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.

Maternal Choline Availability Alters the Localization of p15Ink4B and p27Kip1 Cyclin-Dependent Kinase Inhibitors in the Developing Fetal Rat Brain Hippocampus

Previously we have shown that changes in maternal dietary choline are associated with permanent behavioral changes in offspring. Importantly, in adult male rats, feeding a choline-deficient diet increases the localization of cyclin-dependent kinase inhibitors (CDKIs) in the liver, whereas young adult CDKI knockout mice (p15Ink4B or p27Kip1) exhibit behavioral abnormalities. Thus, maternal dietary choline-CDKI interactions could underlie the changes we observe in fetal hippocampal development and cognitive function in offspring. Here, timed-pregnant rats on embryonic day E12 were fed the AIN-76 diet with varying levels of dietary choline for 6 days, and, on E18, fetal brain sections were collected, and the localization of CDKI proteins was studied using immunohistochemistry and an unbiased image analysis method. In choline-supplemented animals compared to controls, the number of cells with nuclear immunoreactivity for p15Ink4b CDKI protein was decreased 2- to 3-fold in neuroepithelial ventricular zones and adjacent subventricular zones corresponding to the fimbria, primordial dentate gyrus and Ammon’s horn regions in the fetal hippocampus. In contrast, maternal dietary choline deficiency significantly decreased nuclear p15Ink4b immunoreactivity in the neuroepithelial layer of the dentate gyrus. Unlike p15Ink4b, the CDKI protein p27Kip1 was observed almost exclusively in the cytoplasm, though the protein was distributed throughout the proliferating and postmitotic zones in the E18 fetal hippocampus. Maternal dietary choline supplementation decreased the cytoplasmic staining intensity for p27Kip1 throughout the fetal hippocampus compared to control animals. Choline deficiency increased the staining intensity of p27Kip1 throughout the hippocampus in association with increased expression of MAP-1 and vimentin proteins. These results link maternal dietary choline availability to CDKI protein immunoreactivity and commitment to differentiation during fetal hippocampal development.