Phosphatidylcholine biosynthesis via CTP:phosphocholine cytidylyltransferase 2 facilitates neurite outgrowth and branching

Hallmarks of neuronal differentiation are neurite sprouting, extension, and branching. We previously showed that increased expression of CTP:phosphocholine cytidylyltransferase beta2 (CTbeta2), an isoform of a key phosphatidylcholine (PC) biosynthetic enzyme, accompanies neurite outgrowth (Carter, J. M., Waite, K. A., Campenot, R. B., Vance, J. E., and Vance, D. E. (2003) J. Biol. Chem. 278, 44988-44994). CTbeta2 mRNA is highly expressed in the brain. We show that CTbeta2 is abundant in axons of rat sympathetic neurons and retinal ganglion cells. We used RNA silencing to decrease CTbeta2 expression in PC12 cells differentiated by nerve growth factor. In CTbeta2-silenced cells, numbers of primary and secondary neurites were markedly reduced, suggesting that CTbeta2 facilitates neurite outgrowth and branching. However, the length of individual neurites was significantly increased, and the total amount of neuronal membrane was unchanged. Neurite branching of PC12 cells is known to be inhibited by activation of Akt and promoted by the Akt inhibitor LY294002. Our experiments showed that LY294002 increases neurite sprouting and branching in control PC12 cells but not in CTbeta2-deficient cells. CTbeta2 was not phosphorylated in vitro by Akt. However, inhibition of Cdk5 by roscovitine blocked CTbeta2 phosphorylation and reduced neurite outgrowth and branching. These results highlight the importance of CTbeta2 in neurons for promoting neurite outgrowth and branching and represent the first identification of a lipid biosynthetic enzyme that facilitates these functions.     

Involvement of CTP:phosphocholine cytidylyltransferase-β2 in axonal phosphatidylcholine synthesis and branching of neurons

In the brain, phosphatidylcholine (PC) is synthesized by the CDP-choline pathway in which the rate-limiting step is catalyzed by two isoforms of CTP:phosphocholine cytidylyltransferase (CT): CTα and CTβ2. In mice, CTβ2 mRNA is more highly expressed in the brain than in other tissues, and several observations suggest that CTβ2 plays an important role in the nervous system. We, therefore, investigated the importance of CTβ2 for PC synthesis as well as for axon formation, growth and branching of primary sympathetic neurons. We show that in cultured primary neurons nerve growth factor increases the amount of CTβ2, but not CTα, mRNA and protein. The brains of mice lacking CTβ2 had normal PC content despite having 35% lower CT activity than wild-type brains. CTβ2 mRNA and protein are abundant in distal axons of mouse sympathetic neurons whereas CTα mRNA and protein were not detected. Moreover, CTβ2 deficiency in distal axons reduced the incorporation of [(3)H]choline into PC by 95% whereas PC synthesis in cell bodies/proximal axons was unaltered. These data suggest that CTβ2 is the major CT isoform involved in PC synthesis in axons. Axons of CTβ2-deficient sympathetic neurons contained 32% fewer branch points than did wild-type neurons although the number of axons/neuron and the rate of axon extension were the same as in wild-type neurons. We conclude that in distal axons of primary sympathetic neurons CTβ2 is a major contributor to PC synthesis and promotes axon branching, whereas CTα appears to be the major CT isoform involved in PC synthesis in cell bodies/proximal axons.     

Formation of unesterified choline by rat brain

Two preparations of rat brain (ischemic intact brain and homogenized whole brain) formed large amounts of unesterified (free) choline when incubated at 37 degrees C. The accumulation of choline was inhibited by microwave irradiation of brain, or by heating of brain to 50 degrees C, and was maximal at 37 degrees C at pH 7.4-8.5. Choline formation was only observed in subcellular fractions of brain that contained membranes. In homogenates of brain, choline accumulated at a rate exceeding 10 nmol/mg protein per h. There was a significant decrease in brain phosphatidylcholine concentration (of 50 nmol/mg protein) during incubation for 1 h at 37 degrees C. Concentrations of phosphocholine rose (by 2.3 nmol/mg protein), and concentrations of glycerophosphocholine and sphingomyelin did not change during this period. We used radiolabeled phospholipids to trace the fate of phosphatidylcholine and sphingomyelin during incubations of homogenates of brain. Phosphatidylcholine was degraded to form phosphocholine, glycerophosphocholine and free choline. No lysophosphatidylcholine accumulated. Sphingomyelin was degraded to form phosphocholine and a small amount of free choline. Magnesium ions stimulated choline production, while zinc ions were a potent inhibitor. Other divalent cations (calcium, manganese) had little effect on choline accumulation. ATP concentrations in brain homogenates were less than 5 nmol/mg protein (rapidly microwaved brain contained 27 nmol/mg protein). Addition of ATP or ADP to brain homogenates increased ATP concentrations and significantly inhibited choline accumulation. ATP diminished the formation of choline from added phosphatidylcholine, lysophosphatidylcholine, phosphocholine and glycerophosphocholine. The effects of ATP, zinc ion, or magnesium ion upon choline accumulation were not mediated by changes in the rates of utilization of choline for formation of phosphocholine or phosphatidylcholine. In summary, we showed that there was enhanced formation of choline when ATP concentrations within brain were low. This choline was derived, in part, from the degradation of phosphatidylcholine, and we suggest that phospholipase A activity was the primary initiator of choline release from this phospholipid.     

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. © 1997 Wiley-Liss, Inc.     

Role of apoptosis signal-regulating kinase in regulation of the c-Jun N-terminal kinase pathway and apoptosis in sympathetic neurons

We have previously shown that nerve growth factor (NGF) withdrawal-induced death requires the activity of the small GTP-binding protein Cdc42 and that overexpression of an active form of Cdc42 is sufficient to mediate neuronal apoptosis via activation of the c-Jun pathway. Recently, a new mitogen-activated protein (MAP) kinase kinase kinase, apoptosis signal-regulating kinase 1 (ASK1) which activates both the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways and plays pivotal roles in tumor necrosis factor- and Fas-induced apoptosis, has been identified. Therefore, we investigated the role of ASK1 in neuronal apoptosis by using rat pheochromocytoma (PC12) neuronal cells and primary rat sympathetic neurons (SCGs). Overexpression of ASK1-DeltaN, a constitutively active mutant of ASK1, activated JNK and induced apoptosis in differentiated PC12 cells and SCG neurons. Moreover, in differentiated PC12 cells, NGF withdrawal induced a four- to fivefold increase in the activity of endogenous ASK1. Finally, expression of a kinase-inactive ASK1 significantly blocked both NGF withdrawal- and Cdc42-induced death and activation of c-jun. Taken together, these results demonstrate that ASK1 is a crucial element of NGF withdrawal-induced activation of the Cdc42-c-Jun pathway and neuronal apoptosis.     

CHOP is involved in neuronal apoptosis induced by neurotrophic factor deprivation

Neurotrophic factors are essential for the survival of neurons. We found that the endoplasmic reticulum (ER) stress-C/EBP homologues protein (CHOP) pathway to be activated during neurotrophic factor deprivation-induced apoptosis in PC12 neuronal cells and in primary cultured neurons, and this apoptosis was suppressed in the neurons from chop(-/-) mice. In addition, we found that CHOP is expressed in the subventricular zone (SVZ) and striatum of the young adult mouse brain. The number of apoptotic cells in the SVZ decreased in chop(-/-) mice. These results indicate that the ER stress-CHOP pathway plays a role in neuronal apoptosis during the development of the brain.     

Developmental and regional quantitation of glycerophosphorylcholine phosphodiesterase activities in rat brain

The activity of glycerophosphorylcholine cholinephosphodiesterase was quantified in the diencephalon, mesencephalon, cerebral hemispheres, olfactory bulb and cerebellum postnatally for P5 until P70 of rat brain. The initially low activities gradually increase to adult levels by P30. The patterns of regional development are reminescent of those previously described for choline acetyltransferase activity. It is suggested that these may be functionally linked in neuronal cells. The activity of glycerophosphorylcholine phosphocholine phosphodiesterase was also determined and found to be similar although only one half as active as the enzyme liberating choline. The present experiments show that both the GPC phosphocholine phosphodiesterase and the GPC choline phosphodiesterase are regionally and developmentally regulated in rat brain.     

Prevention of peroxynitrite-induced apoptosis of motor neurons and PC12 cells by tyrosine-containing peptides

Although peroxynitrite stimulates apoptosis in many cell types, whether peroxynitrite acts directly as an oxidant or the induction of apoptosis is because of the radicals derived from peroxynitrite decomposition remains unknown. Before undergoing apoptosis because of trophic factor deprivation, primary motor neuron cultures become immunoreactive for nitrotyrosine. We show here using tyrosine-containing peptides that free radical processes mediated by peroxynitrite decomposition products were required for triggering apoptosis in primary motor neurons and in PC12 cells cultures. The same concentrations of tyrosine-containing peptides required to prevent the nitration and apoptosis of motor neurons induced by trophic factor deprivation and of PC12 cells induced by peroxynitrite also prevented peroxynitrite-mediated nitration of motor neurons, brain homogenates, and PC12 cells. The heat shock protein 90 chaperone was nitrated in both trophic factor-deprived motor neurons and PC12 cells incubated with peroxynitrite. Tyrosine-containing peptides did not affect the induction of PC12 cell death by hydrogen peroxide. Tyrosine-containing peptides should protect by scavenging peroxynitrite-derived radicals and not by direct reactions with peroxynitrite as they neither increase the rate of peroxynitrite decomposition nor decrease the bimolecular peroxynitrite-mediated oxidation of thiols. These results reveal an important role for free radical-mediated nitration of tyrosine residues, in apoptosis induced by endogenously produced and exogenously added peroxynitrite; moreover, tyrosine-containing peptides may offer a novel strategy to neutralize the toxic effects of peroxynitrite.     

Regulation of phosphatidylcholine biosynthesis by mitogenic growth factors

Phosphatidylcholine (PC) biosynthesis in cultured 3T3 fibroblasts was increased in varying degrees by these mitogenic growth factors: fetal bovine serum, insulin, 12-O-tetradecanoylphorbol-13-acetate, epidermal growth factor, vasopressin, fibroblast growth factor and insulin-like growth factors I and II. PC synthesis was increased 2-4-fold by 10% serum, up to 4-fold by growth factors alone, and up to 8-fold by combinations of two or more growth factors. Single growth factors had no effect on the incorporation of [3H]choline into the acid-soluble precursors of PC, while serum or combinations of two or more mitogens could increase the incorporation of [3H]choline into acid-soluble material by up to 2-fold. Serum was shown to increase choline phosphorylation, choline kinase activity and the size of the phosphocholine pool. These data were utilized to calculate the radioactive specific activity of phosphocholine. Serum did not increase phosphocholine specific activity above control values; thus the increased incorporation of labelled choline into PC after serum stimulation resulted from increased PC synthesis and not from a simple change in specific activity of precursor phosphocholine.     

Effect of cAMP on choline uptake and phosphatidylcholine biosynthesis in cultured chick heart cells

The effect of an analogue of cAMP on the uptake and metabolism of choline in the heart was studied in isolated cardiac cells. The cells were obtained from 7-day-old chick embryos and maintained in culture. The effects of cAMP were studied using the dibutyryl cAMP analogue and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. After a 2-h incubation with [3H]choline, about 85% of the label was recovered in phosphocholine, with most of the rest in phospholipid. During a subsequent chase incubation, [3H]phosphocholine was transferred to phosphatidylcholine with little accumulation in CDP-choline. This suggests the rate-limiting step for the conversion of phosphocholine to phosphatidylcholine in these cells is the synthesis of CDP-choline. cAMP decreased the incorporation of choline into phosphatidylcholine, but did not change the flux of metabolites through the step catalyzed by CTP:phosphocholine cytidylyltransferase. cAMP had little effect on choline uptake at low (1-25 microM) extracellular choline concentrations, but significantly (p less than 0.05) decreased choline uptake at higher (37.5-50 microM) extracellular choline concentrations. Thus, cardiac cells take up and metabolize choline to phosphocholine, with CTP:phosphocholine cytidylyltransferase being the rate-limiting step in phosphatidylcholine biosynthesis. cAMP decreases [3H]choline uptake and its subsequent incorporation into phosphocholine and phospholipid. However, the metabolism of choline within the cell is unaffected.     

Biosynthesis of phosphatidylcholine from a phosphocholine precursor pool derived from the late endosomal/lysosomal degradation of sphingomyelin

Previous studies suggest that the steps of the CDP- choline pathway of phosphatidylcholine synthesis are tightly linked in a so-called metabolon. Evidence has been presented that only choline that enters cells through the choline transporter, and not phosphocholine administered to cells by membrane permeabilization, is incorporated into phosphatidylcholine. Here, we show that [(14)C]phosphocholine derived from the lysosomal degradation of [(14)C]choline-labeled sphingomyelin is incorporated as such into phosphatidylcholine in human and mouse fibroblasts. Low density lipoprotein receptor-mediated endocytosis was used to specifically direct [(14)C]sphingomyelin to the lysosomal degradation pathway. Free labeled choline was not found either intracellularly or in the medium, not even when the cells were energy-depleted. Deficiency of lysosomal acid phosphatases in mouse or alkaline phosphatase in human fibroblasts did not affect the incorporation of lysosomal [(14)C]sphingomyelin-derived [(14)C]phosphocholine into phosphatidylcholine, supporting our finding that phosphocholine is not degraded to choline prior to its incorporation into phosphatidylcholine. Inhibition studies and analysis of molecular species showed that exogenous [(3)H]choline and sphingomyelin-derived [(14)C]phosphocholine are incorporated into phosphatidylcholine via a common pathway of synthesis. Our findings provide evidence that, in fibroblasts, phosphocholine derived from sphingomyelin is transported out of the lysosome and subsequently incorporated into phosphatidylcholine without prior hydrolysis of phosphocholine to choline. The findings do not support the existence of a phosphatidylcholine synthesis metabolon in fibroblasts.     

Choline release and inhibition of phosphatidylcholine synthesis precede excitotoxic neuronal death but not neurotoxicity induced by serum deprivation

N-Methyl-d-aspartate (NMDA) receptor overactivation has been proposed to induce excitotoxic neuronal death by enhancing membrane phospholipid degradation. In previous studies, we have shown that NMDA releases choline and reduces membrane phosphatidylcholine in vivo. We now observed that glutamate and NMDA induce choline release in primary neuronal cortical cell cultures. This effect is Ca(2+)-dependent and is blocked by MK-801 ((+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate). In cortical neurons, the NMDA receptor-mediated choline release precedes excitotoxic cell death but not neuronal death induced by either osmotic lysis or serum deprivation. Glutamate, at concentrations that release arachidonic acid, does not release choline in cerebellar granule cells, unless these cells are rendered susceptible to excitotoxic death by energy deprivation. The NMDA-evoked release of choline is not mediated by phospholipases A(2) or C. Moreover, NMDA does not activate phospholipase D in cortical cells. However, NMDA inhibits incorporation of [methyl-(3)H]choline into both membrane phosphatidylcholine and sphingomyelin. These results show that the increase in extracellular choline induced by NMDA receptor activation is directly related with excitotoxic cell death and indicate that choline release is an early event of the excitotoxic process produced by inhibition of phosphatidylcholine synthesis and not by activation of membrane phospholipid degradation.     

Regulation of miR-34 Family in Neuronal Development

Differentiation of neural stem cells (NSC’s) to mature and functional neurons requires coordinated expression of mRNA, microRNAs (miRNAs) and regulatory proteins. Our earlier unbiased miRNA profiling studies have identified miR-200, miR-34 and miR-221/222 as maximally up-regulated miRNA families in differentiating PC12 cells and demonstrated the capability of miR-200 family in inducing neuronal differentiation (J. Neurochem, 2015, 133, 640–652). In present study, we have investigated role of miR-34 family in neuronal differentiation and identified P53 as mediator of nerve growth factor (NGF) induced miR-34a expression in differentiating PC12 cells. Our studies have shown that NGF induced miR-34a, arrests proliferating PC12 cells to G1 phase, which is pre-requisite for neuronal differentiation. Our studies have also shown that increased expression of miR-34a controls the P53 level in differentiated PC12 cells in feedback inhibition manner, which probably prevents differentiated cells from P53 induced apoptosis. Expression profiling of miR-34 family in different neuronal, non-neuronal and developing cells have identified differentiated and aged brain cells as richest source of miR-34, which also indicates that higher expression of miR-34 family helps in maintaining the mature neurons in non-proliferative stage. In conclusion, our studies have shown that miR-34 is brain enriched miRNA family, which up-regulates with neuronal maturation and brain ageing and co-operative regulation of P53 and miR-34a helps in neuronal differentiation by arresting cells in G1 phase.     

Carboxyl-terminal fragment of Alzheimer's APP destabilizes calcium homeostasis and renders neuronal cells vulnerable to excitotoxicity.

Numerous lines of evidence indicate that some of the neurotoxicity associated with Alzheimer's disease (AD) is due to proteolytic fragments of the amyloid precursor protein (APP). Most research has focused on the amyloid beta peptide (Abeta). However, the possible role of other cleaved products of APP is less clear. We have previously shown that a recombinant carboxy-terminal 105 amino acid fragment (CT 105) of APP induced strong nonselective inward currents in XENOPUS: oocyte; it also revealed neurotoxicity in PC12 cells and primary cortical neurons, blocked later phase of long-term potentiation in rat hippocampus in vivo, and induced memory deficits and neuropathological changes in mice. We report here that the pretreatment with CT 105 for 24 h at a 10 microM concentration increases intracellular calcium concentration by about twofold in SK-N-SH and PC 12 cells, but not in U251 cells, originated from human glioblastoma. In addition, the calcium increase and toxicity induced by CT 105 were reduced by cholesterol and MK 801 in SK-N-SH and PC 12 cells, whereas the toxicity of Abeta(1-42) was attenuated by nifedipine and verapamil. CT 105 rendered SK-N-SH cells and rat primary cortical neurons more vulnerable to glutamate-induced excitotoxicity. Also, conformational studies using circular dichroism experiments showed that CT 105 has approximately 15% of beta-sheet content in phosphate buffer and aqueous 2,2, 2-trifluoroethanol solutions. However, the content of beta-sheet conformation in dodecyl phosphocholine micelle or in the negatively charged vesicles, is increased to 22%-23%. The results of this study showed that CT 105 disrupts calcium homeostasis and renders neuronal cells more vulnerable to glutamate-induced excitotoxicity, and that some portion of CT 105 has partial beta-sheet conformation in various environments, which may be related to the self-aggregation and toxicity. This may be significantly possibly involved in inducing the neurotoxicity characteristic of AD.     

RAIDD aggregation facilitates apoptotic death of PC12 cells and sympathetic neurons

In human cell lines, the caspase 2 adaptor RAIDD interacts selectively with caspase 2 through its caspase recruitment domain (CARD) and leads to caspase 2-dependent death. Whether RAIDD induces such effects in neuronal cells is unknown. We have previously shown that caspase 2 is essential for apoptosis of trophic factor-deprived PC12 cells and rat sympathetic neurons. We report here that rat RAIDD, cloned from PC12 cells, interacts with rat caspase 2 CARD. RAIDD overexpression induced caspase 2 CARD- and caspase 9-dependent apoptosis of PC12 cells and sympathetic neurons. Apoptosis correlated with the formation of discrete perinuclear aggregates. Both death and aggregates required the expression of full-length RAIDD. Such aggregates may enable more effective activation of caspase 2 through close proximity. Following trophic deprivation, RAIDD overexpression increased death and aggregate formation. Therefore, RAIDD aggregation is important for its death-promoting effects and may play a role in trophic factor withdrawal-induced neuronal apoptosis.     

Bombesin and phorbol ester stimulate phosphatidylcholine hydrolysis by phospholipase C: evidence for a role of protein kinase C

Bombesin caused a marked stimulation of 32Pi into phosphatidylinositol (PI), with no apparent lag, and into phosphatidylcholine (PC), after a lag of about 20 min. Stimulation was blocked by the bombesin receptor antagonist, [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P, indicating that the effects on both PI and PC were mediated through the same receptor. The tumor-promoting phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) and dioctanoylglycerol (diC8) both directly activate protein kinase C and in this report were shown to stimulate 32Pi incorporation into PC but not into Pl. In addition, TPA stimulated the release of [3H]choline and [3H]phosphocholine and the accumulation of [3H]diacyglycerol from prelabelled cells. These results strongly suggest that TPA activates a phospholipase C specific for PC. Pretreatment of cells with phorbol-12, 13-dibutyrate (PDBu) for 24 h depleted cellular protein kinase C activity and inhibited the ability of TPA to induce these effects suggesting a direct involvement of protein kinase C. Similarly the bombesin stimulation of 32Pi into PC and of [3H]choline and [3H]phosphocholine release was inhibited by PDBu pretreatment. DiC8 and, to a lesser extent, TPA stimulated the translocation of CTP:phosphocholine cytidylytransferase from the cytosolic to the particulate fraction. DiC8 also stimulated this translocation in cells depleted of protein kinase C. It was concluded that both bombesin and TPA activated protein kinase C leading to activation of a phospholipase C specific for PC.     

Withdrawal of Survival Factors Results in Activation of the JNK Pathway in Neuronal Cells Leading to Fas Ligand Induction and Cell Death

The JNK pathway modulates AP-1 activity. While in some cells it may have proliferative and protective roles, in neuronal cells it is involved in apoptosis in response to stress or withdrawal of survival signals. To understand how JNK activation leads to apoptosis, we used PC12 cells and primary neuronal cultures. In PC12 cells, deliberate JNK activation is followed by induction of Fas ligand (FasL) expression and apoptosis. JNK activation detected by c-Jun phosphorylation and FasL induction are also observed after removal of either nerve growth factor from differentiated PC12 cells or KCl from primary cerebellar granule neurons (CGCs). Sequestation of FasL by incubation with a Fas-Fc decoy inhibits apoptosis in all three cases. CGCs derived from gld mice (defective in FasL) are less sensitive to apoptosis caused by KCl removal than wild-type neurons. In PC12 cells, protection is also conferred by a c-Jun mutant lacking JNK phosphoacceptor sites and a small molecule inhibitor of p38 mitogen-activated protein kinase and JNK, which inhibits FasL induction. Hence, the JNK-to-c-Jun-to-FasL pathway is an important mediator of stress-induced neuronal apoptosis.     

Apoptosis in human embryo development: 3. Fas-induced apoptosis in brain primary cultures

Fas (APO-1/CD95) is an important apoptotic mediator for both immune and nervous systems. In the present study, we have investigated the expression and function of Fas in human embryonic/fetal brain primary cultures from 12 human embryos and fetuses with gestational ages between 5 to 22 weeks. Anti-Fas fluorescent antibody was used for labeling of Fas positive cells and for quantitation of Fas expression in brain cultures. To demonstrate that Fas receptor is functional in human embryonic/fetal brain cells, anti-Human-Fas monoclonal antibody (0.5 μg/ml) was used to induce apoptosis in brain primary cultures. Apoptosis was investigated by flow-cytometry and fluorescent microscopy using TUNEL and annexin V labeling. Fas was found to be expressed in the embryonic/fetal human primary brain cultures, on neuronal and glial cells or their precursors, varying with gestational ages. Cross-linking of Fas induced apoptosis in brain cultures indicating that Fas receptor functions as a death receptor. We also showed that cell death triggered through Fas receptor was caspase dependent, hence it was blocked by a selective caspase-8 inhibitor (IETD-fmk).These results suggest that Fas is involved in neuronal apoptosis in the developing human brain.     

Turnover of phosphatidylcholine in Saccharomyces cerevisiae. The role of the CDP-choline pathway

The regulation of phosphatidylcholine degradation as a function of the route of phosphatidylcholine (PC) synthesis and changing environmental conditions has been investigated in the yeast Saccharomyces cerevisiae. In the wild-type strains studied, deacylation of phosphatidylcholine to glycerophosphocholine is induced when choline is supplied to the culture medium and, also, when the culture temperature is raised from 30 to 37 degrees C. In strains bearing mutations in any of the genes encoding enzymes of the CDP-choline pathway for phosphatidylcholine biosynthesis (CKI1, choline kinase; CPT1, 1, 2-diacylglycerol choline phosphotransferase; PCT1, CTP:phosphocholine cytidylyltransferase), no induction of phosphatidylcholine turnover and glycerophosphocholine production is seen in response to choline availability or elevated temperature. In contrast, the induction of phosphatidylcholine deacylation does occur in a strain bearing mutations in genes encoding enzymes of the methylation pathway for phosphatidylcholine biosynthesis (i.e. CHO2/PEM1 and OPI3/PEM2). Whereas the synthesis of PC via CDP-choline is accelerated when shifted from 30 to 37 degrees C, synthesis of PC via the methylation pathway is largely unaffected by the temperature shift. These results suggest that the deacylation of PC to GroPC requires an active CDP-choline pathway for PC biosynthesis but not an active methylation pathway. Furthermore, the data indicate that the synthesis and turnover of CDP-choline-derived PC, but not methylation pathway-derived PC, are accelerated by the stress of elevated temperature.