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Address correspondence to Erhard Bieberich, Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th Street, Room CB-2803, Augusta, GA 30912. Tel.: (706) 721-9113. Fax: (706) 721-8685. email: ebieberich{at}mail.mcg.edu
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Abstract |
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Key Words: embryonic stem cells; neuronal differentiation; neuroprogenitor; sphingolipid; nestin
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Introduction |
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According to a current model, apoptosis of neuronal stem cells may occur very soon after cell division, leaving one daughter cell committed to cell death, whereas the other one may further proliferate or differentiate (Kuan et al., 2000; Sommer and Rao, 2002). This asymmetric model of apoptosis during neural differentiation implies that cell death or survival is regulated immediately on cell division of stem cells. Most recently, asymmetric distribution of the Notch inhibitor Numb to one daughter cell during mitosis of neural precursor cells has been suggested to regulate cell death in Drosophila and mammals (Orgogozo et al., 2002; Shen et al., 2002). We hypothesized that asymmetric cell death of mammalian neural progenitors (NPs) may also result from the unequal distribution of pro- or anti-apoptotic factors to the daughter cells during cell division. We tested this hypothesis by analyzing the expression of several pro- or anti-apoptotic proteins involved in ceramide-induced apoptosis of differentiating embryonic stem (ES) cells and their correlation with cell division and death.
The observation that ceramide and PAR-4 are concurrently elevated during the peak time of apoptosis in embryonic mouse brain (Bieberich et al., 2001) prompted us to determine the function of ceramide and PAR-4 in the regulation of NP cell death. Mouse ES cells have been shown to differentiate in culture into neurons and glial cells by recapitulating the stages of neuronal differentiation that occur in vivo (Fraichard et al., 1995; Mayer-Proschel et al., 1997). In particular, the formation of NP cells is a critical stage of commitment and differentiation into neuronal and glial cells. In ES cells, this stage occurs during serum deprivation on embryoid body (EB) formation, and during the expansion of EB-derived cells in serum-free medium plus fibroblast growth factor 2 (FGF-2; Hancock et al., 2000). In embryonic mouse brain, these stages probably correspond to populations of differentiating neural stem cells in the subventricular/ventricular zone between E12 and E18 (Hatten 1999). Accordingly, in vitro neural differentiation of ES cells by serum deprivation is a valid model for the functional correlation of ceramide and PAR-4 elevation with induction of apoptosis in both, in vitro differentiating ES cells and neural stem cells in embryonic mouse brain.
To define the molecular mechanisms underlying ceramide-induced apoptosis in NP cells, we have analyzed the expression levels and patterns of ceramide and PAR-4 in NP cells derived from ES cells. We have used high performance TLC (HPTLC) of lipid extracts of differentiating ES cells to measure ceramide levels during differentiation. The intracellular distribution of ceramide was detected using an antibody that has been used for specific immunostaining of ceramide in fixed cells (Grassme et al., 2001). Immunofluorescence microscopy of NP cells revealed that PAR-4, ceramide, and the intermediate filament protein nestin are asymmetrically distributed during cell division. The coexpression of PAR-4 and ceramide was concurrent with TUNEL staining for apoptosis. The other daughter cell that did not express PAR-4 was nestin positive and was not apoptotic. Thus, asymmetric distribution of PAR-4 may regulate ceramide-induced apoptosis during the proliferation and differentiation of stem cells.
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Results |
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To suppress PAR-4 expression, ES cells at the NP1 stage were transfected with a PAR-4specific morpholino phosphorodiamidate antisense oligonucleotide (morpholino) that was designed on the basis of a previously published antisense oligonucleotide sequence (Guo et al., 1998, 2001). Morpholinos have been shown to avoid many of the pitfalls associated with conventional antisense oligonucleotides, and have been successfully used for transfection of embryos and cultured cells (Morcos, 2001). Fig. 5 A (left) shows NP cells 48 h after transfection with a standard morpholino provided as negative control. The number of TUNEL-stained cells in the negative control was equivalent to that found with untransfected cells (35%, see Fig. 3 B), indicating that the degree of apoptosis was not affected due to any unspecific effect of the morpholino. This result was consistent with the observation that the expression level of PAR-4 in untransfected and control morpholino-transfected NP cells was the same (Fig. 5 B, lane 1 and lane 2). Incubation of control morpholino-transfected NP cells with 80 µM S18 increased the number of apoptotic cells to that found with S18-treated, untransfected cells (>80%). However, transfection of NP cells with a PAR-4specific antisense morpholino reduced S18-induced apoptosis to a level <30% (Fig. 5 A, right). Consistently, transfection with the PAR-4 antisense morpholino suppressed the expression level of PAR-4 to 25% of that detected in untransfected cells (Fig. 5 B, lane 3).
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Apoptosis is induced by simultaneous up-regulation of PAR-4 and ceramide biosynthesis
To identify the genes involved in the regulation of apoptosis in differentiating ES cells, we have determined the temporal expression pattern of pro- or anti-apoptotic proteins and ceramide biosynthesis/metabolism during EB and NP formation. Fig. 6 A shows that PAR-4 expression was significantly elevated at the EB8 and NP2 stages (Fig. 6 A, lane 1 and lane 2). By the first day of neural differentiation (D1), the PAR-4 level dropped to <20% of that at NP2 (Fig. 6 A, lane 2 and lane 3). The peak time of PAR-4 expression at the NP2 stage was concurrent with caspase 3 activation and increased proliferating cell nuclear antigen (PCNA) levels (Fig. 6 A, lane 2). PCNA is a marker for mitotic cells (Chan et al., 2002), indicating that PAR-4 elevation and caspase 3 activation is predominant at stages with a large number of proliferating stem cells. The expression of PKC did not change during neuronal differentiation. PKC
activity is inhibited by ceramide-enhanced binding of PAR-4, leading to an up-regulation of the caspase 9 and caspase 3dependent apoptotic pathway (Diaz-Meco et al., 1996; Mattson, 2000). We confirmed the participation of caspase 9 in ceramide-induced apoptosis of differentiating ES cells by the observation that preincubation with the cell-permeable caspase 9 inhibitor peptide LEHD-CHO suppressed apoptosis that was inducible with S18 or natural ceramide isolated from ES cells. Upstream regulators of caspase 3, in particular, anti-apoptotic b-cell lymphoma 2 (Bcl-2) and pro-apoptotic Bcl-2 antagonist of death (Bad) were inversely regulated, suggesting activation of caspase 3 via the mitochondrial death pathway (Fig. 6 A). However, a high degree of Bad phosphorylation was detectable at NP2 (Fig. 6 A, lane 2) indicating the presence of both apoptotic and anti-apoptotic signaling at this stage. The expression of Bad dropped and that of Bcl-2 increased during D1 and D4, consistent with lower levels of caspase 3 activation and apoptosis at these differentiation stages (Fig. 6 A, lane 3 and lane 4).
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Asymmetric distribution of PAR-4 and nestin during mitosis of neuronal stem cells
The apparent paradoxical elevation of both the proliferation marker PCNA and the pro-apoptotic, active caspase 3 at the EB8 and NP2 stages of ES cell differentiation prompted us to examine the patterns of mitosis and apoptosis in individual cells by immunofluorescence microscopy for mitosis/apoptosis markers and BrdU labeling. We also analyzed whether mitosis or apoptosis is predominant in NP cells by immunofluorescence staining for nestin. As shown in Table I and Fig. 7 A, simultaneous staining by TUNEL assay (green) and antibodies against nestin (red) and PCNA (far-red, pseudo-colored in pink) revealed that the major portion (92%) of TUNEL-positive cells were nestin negative (see also Fig. 4). Most of the TUNEL-positive cells (75%) expressed PCNA as well (overlay yields white-colored cells in Fig. 7 A), indicating that apoptosis followed immediately after mitosis or during the attempt to enter the next mitotic cycle. However, no TUNEL signal was detected if cells coexpressed PCNA and nestin. These observations prompted us to look specifically for the distribution of pro-apoptotic signals in proliferating cells, in particular for the expression of PAR-4 and ceramide. In double-staining experiments for TUNEL and one additional marker, ceramide or PAR-4 is expressed in TUNEL-positive as well as TUNEL-negative cells (Table I). The majority of the TUNEL-positive cells expressed PAR-4 (94%), ceramide (98%), or PCNA (75%), whereas TUNEL-negative cells showed a lower frequency for PAR-4 (27%), ceramide (34%), or PCNA (45%) expression (Table I). In double-staining experiments for two markers, PAR-4 and ceramide are independently distributed (31% observed vs. 24% expected frequency for the expression of both ceramide and PAR-4) within the total cell population. However, in triple-staining experiments, 97% of the TUNEL-positive cells show PAR-4 and ceramide expression, whereas less than 2% of the TUNEL-negative cells are stained for PAR-4 as well as ceramide. Together, these results indicate that the expression of both PAR-4 and ceramide is required to induce apoptosis in proliferating (PCNA positive) stem cells or NP cells. This assumption was also supported by immunostaining for BrdU incorporation in apoptotic cells. Fig. 7 B shows that in differentiating ES cells at the NP2 stage, apoptosis is observed in 70% of cells within 5 h after BrdU labeling. Hence, in differentiating ES cells at the NP stage, apoptosis (activation of caspase 3) rapidly follows mitosis (BrdU incorporation).
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Discussion |
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In cultures of NP cells derived from ES cells, we found that the expression of PAR-4 and nestin is largely segregated to two separate populations of progenitors. Nestin(+)/PAR-4(-) cells are far less prone to apoptosis induced by high levels of intracellular ceramide, whereas cells undergoing apoptosis are almost always nestin(-)/PAR-4(+). The expression of high levels of PAR-4 in an apoptotic subpopulation is consistent with previous reports demonstrating that overexpression of PAR-4 reduces WT1- or nuclear factor kappa B (NFB)mediated Bcl-2 expression, thus suppressing the cells' self-defense mechanism against ceramide-induced apoptosis (Camandola and Mattson, 2000; Cheema et al., 2003). The critical role of NF
B for cell survival has been shown by the observation that central neuron survival relies on the constitutive activity of NF
B (Bhakar et al., 2002). It has also been shown that elevation of PAR-4 underlies neuronal cell death in several neurodegenerative diseases (Guo et al., 1998; Xie et al., 2001). To our surprise, in cells that express both PAR-4 and nestin, the two proteins are strictly sequestered to different parts of the cell. This sequestration occurs already during mitotic cell division of the parental cells, resulting in one daughter cell that is predominantly nestin-positive, while the other one contains mainly PAR-4, but no (or only low) amounts of nestin. This reveals a novel localization of a pro-apoptotic signal to one of the daughter cells resulting from stem or NP cell division. The asymmetric distribution of PAR-4 in the nestin-negative daughter cells may be a mechanism to regulate the number of differentiating cells produced from mitotically active progenitor/stem cells. At present, it cannot be decided whether the asymmetric distribution results from asymmetric inheritance of nestin and PAR-4 that have already expressed before cell division, or whether it arises from a distinct gene expression in each daughter cell during cell division.
Based on these observations, we suggest a model for asymmetric cell division, apoptosis, and differentiation of neuronal stem cells, shown in Fig. 10. Differentiating stem cells up-regulate the expression of nestin, ceramide, and PAR-4 before or during cell division. Ceramide and PAR-4 elevation at these stages is most likely caused by up-regulation of gene expression for PAR-4 and SPT subunit 1 (SPT1) in EB8. This indicates regulation of de novo ceramide biosynthesis and PKC activity by the respective regulatory proteins, but not by basal enzyme activities. During mitosis, ceramide is evenly sequestered to the two daughter cells, whereas PAR-4 and nestin are asymmetrically distributed. The nestin(-)/PAR-4(+) daughter cell undergoes ceramide-induced apoptosis, whereas the nestin(+)/PAR-4(-) daughter cell may again divide or further differentiate into a neuronal or glial precursor cell. At this point, rapid passage of the mitotic cycle is desirable in order to sequester PAR-4 to one daughter cell and to avoid abortive mitosis before accomplishment of cell division (Liu and Greene, 2001).
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Materials and methods |
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In vitro neuronal differentiation of ES cells
In vitro neuronal differentiation of mouse ES cells (ES-J1, ES-D3) followed a serum deprivation protocol (Okabe et al., 1996; Hancock et al., 2000). In brief, ES cells were grown on -irradiated feeder fibroblasts for 4 d in knockout DME/15% knockout serum replacement, supplemented with ESGRO (LIF). ES cells were then passaged onto gelatin-coated tissue culture dishes without feeder fibroblasts and incubated for 4 d in knockout DME/15% heat-inactivated ES-qualified FBS, supplemented with 1,000 U/ml ESGRO (LIF). On trypsinization, ES cells were transferred to bacterial culture dishes without gelatin in order to allow EB formation. EBs were incubated for 4 d in knockout DME/10% heat-inactivated ES-qualified FBS. We refer to this stage as EB4. On the fifth day, floating and loosely attached EBs were rinsed off, transferred to tissue culture dishes, and incubated overnight in knockout DME, 10% heat-inactivated ES-qualified FBS to the allow the EBs to attach to the dish. Neuronal differentiation due to serum deprivation was induced by cultivation for 3 d in DME/Ham's F12 (50/50), 1x N2 supplement. We refer to this stage as EB8. Serum-deprived EBs were then trypsinized or treated with a nonenzymatic cell dissociation solution, plated on poly-L-ornithine/laminincoated tissue culture dishes and grown for 4 d in DME/Ham's F12 (50/50), supplemented with 1x N2 and 10 ng/ml FGF-2. We refer to this incubation period as the neuroprogenitor (NP) stage due to the selective expansion of NP cells in the FGF-2 containing serum-free medium. We refer to NP cells grown for 48 h after replating of trypsinized EBs as the NP2 stage. On the fifth day of NP formation, the medium is changed to neurobasal, 5% heat-inactivated FBS, and cells are incubated for another 7 d. During this time, NP cells become fully differentiated to glial cells and neurons. We refer to 24 or 96 h of differentiation as the D1 or D4 stages, respectively (see Fig. 1 for diagram of differentiation protocol).
Ceramide analysis and preparation of ceramide-containing medium
The extraction and quantitative determination of ceramide by HPTLC followed a standard protocol as described previously (Bieberich et al., 2001). Lipids were stained with 3% cupric acetate in 8% phosphoric acid for quantification by comparison with various amounts of standard lipids. For quantitative determination of ceramide using the DAG kinase assay according to Signorelli and Hannun (2002). For incubation of FB1- or myriocin-treated (ceramide-depleted) differentiating ES cells with natural ceramide, 50 mg EBs or NP cells were resuspended in 500 µl of water, and after phase separation with 500 µl of CHCl3/CH3OH (1:1, vol/vol), the neutral lipids recovered from the lower phase. The neutral lipids were evaporated to dryness with a gentle stream of nitrogen and were redissolved in 1 ml CHCl3. The solution was applied to a 0.5-g silicic acid gel column, and fatty acids and cholesterol were washed out with another 15 ml CHCl3 (Dasgupta and Hogan, 2001). The ceramide fraction was then eluted with 20 ml of CHCl3/acetone (9:1, vol/vol), evaporated to dryness, and the residue (6 nmol of ceramide) dissolved in 20 µl ethanol containing 2% dodecane (vol/vol). 5-µl aliquots were mixed with 1 ml medium yielding a final concentration of 1.5 µM natural ceramide for induction of apoptosis (Ji et al., 1995).
BrdU labeling, immunofluorescence microscopy, and TUNEL assay
Differentiating ES cells at the EB8 stage were dissociated and grown for 24 h on laminin/ornithin-coated coverslips (NP1 stage) in DME/Ham's F12 (50/50), supplemented with 1x N2 and 20 ng/ml FGF-2. Cells were incubated for 3 h with 10 µM BrdU, and the TUNEL assay or immunostainings were performed after 5 h of incubation. For immunostaining, cells were fixed with 4% PFA in PBS. Fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min at RT, and immunostaining was performed as described previously (Bieberich et al., 2001). The nuclei were stained by treatment with 2 µg/ml Hoechst 33258 in PBS for 30 min at RT. Apoptotic nuclei were stained using the fluorescein FragELTM TUNEL assay according to instructions of the manufacturer (Oncogene Research Products).
Statistical analysis
Antigen-specific immunostaining was quantified by counting cells that showed signals twofold or more above background fluorescence. Cell counts were performed in five areas of 200 cells that were obtained from three independent immunostaining reactions. A Chi-square test with one degree of freedom was applied for the statistical analysis of the distribution of two immunostained antigens. The first null hypothesis (H01) to be refuted was that the two antigens were independently distributed within the total cell population (mean of 200 cells in five counts). The expected frequency for double staining was the frequency product for immunostaining of A or B in the total population, f(A and B) = f(A) x f(B). The second null hypothesis (H02) to be refuted was that the frequency of antigen B in the subpopulation A was identical to its frequency in the total population, f(B in A) = f(B in A + B).
RNA preparation and RT-PCR
Total RNA was prepared from differentiating stem cells using the TRIzol® method according to the manufacturer's protocol (Life Technologies). An aliquot (0.61.0 µg RNA) was used for RT-PCR with the ThermoScriptTM RT-PCR system following the supplier's instructions (Invitrogen). PCR was performed by applying 35 cycles with various amounts of first-strand cDNA template (equivalent to 0.050.2 µg of RNA) and 20 pmol of sense and antisense oligonucleotide primer. The following oligonucleotide primer sequences and annealing temperatures were used: PAR-4 (sense, 5'-ccagcgccaggaaaggcaaag-3'; antisense, 5'-ctaccttgtcagctgcccaacaac-3'; 61°C), PKC (sense, 5'-agccacgccgtttggaaagg-3'; antisense, 5'-acactttattcctcagggcattacacg-3'; 58°C), SPT1 (sense, 5'-gctaacatggagaatgcactc-3'; antisense, 5'-cttcctccgtctgctccac-3'; 53°C), and GAPDH (sense, 5'-gaaggtgaaggtcggagtcaacg-3'; antisense, 5'-ggtgatgggatttccattgatgacaagc-3'; 58°C). The amount of template from each sample was adjusted until PCR yielded equal intensities of amplification for GAPDH.
Construction of PAR-4-RFP cDNA and transfection of EB-derived cells
For construction of PAR-4-RFP cDNA, RT-PCR was performed with the oligonucleotide primer pair sense 5'-atggcgaccggcggctatcg-3' and antisense 5'-ctaccttgtcagctgcccaacaac-3' using the first-strand cDNA generated from EBs (EB8 stage) as template for the amplification reaction. The primers were endowed with the restriction enzyme cleaving sites Eco47III (sense) and SalI (antisense) for ligation of the PAR-4specific amplification product into the multiple cloning site of HcRFP, a vector that encodes a far-redshifted variant of red fluorescent protein (CLONTECH Laboratories, Inc.). Differentiating ES cells at the NP1 stage were transfected with the PAR-4-RFP construct using the LipofectAMINETM 2000 procedure according to the manufacturer's instructions (Invitrogen). The transfected cells were incubated in DME/Ham's F12 (50/50), supplemented with 1x N2 and 20 ng/ml FGF-2, and the TUNEL assay or immunostainings were performed after 48 h of incubation and NP formation. For depletion of ceramide, differentiating ES cell were incubated with 25 µM FB1 or 50 nM myriocin 48 h before transfection with the PAR-4-RFP vector, and the inhibitor was maintained in the medium throughout the post-transfection period. For induction of apoptosis, the novel ceramide analogue S18 (40100 µM), N-acetyl sphingosine (C2-ceramide; 1030 µM), or natural ceramide isolated from EBs (0.51.5 µM, for preparation see section on ceramide analysis) was added 48 h after transfection with the PAR-4-RFP cDNA, and the degree of apoptosis was quantified after 1520 h using TUNEL assays with PFA-fixed cells as described in the section on BrdU labeling.
Morpholino antisense knockdown of endogenous PAR-4
Endogenous expression of PAR-4 was suppressed by transfection of differentiating ES cells at the NP1 stage with 2 nmol (per well in 24-well plates) of the morpholino-based antisense oligonucleotide 5'-cgatagccgccggtcgccatgttcc-3' (for sequence see Guo et al., 1998, 2001) following the instructions of the manufacturer (GeneTools) in 0.5 ml serum-free DME/Ham's F-12 and 1x N2 supplemented with 20 ng/ml FGF-2. For induction of apoptosis, the novel ceramide analogue S18 (40100 µM), N-acetyl sphingosine (C2-ceramide; 1030 µM), or natural ceramide isolated from EBs (0.51.5 µM, for preparation see section on ceramide analysis) was added 48 h after transfection with the anti-PAR-4 morpholino, and the degree of apoptosis was quantified after 1520 h using TUNEL assays with PFA-fixed cells as described in the section on BrdU labeling.
Protein isolation and immunoblotting
The amount of protein was determined after a modified Folin phenol reagent (Lowry) assay as described previously (Wang and Smith, 1975). Protein extracted with detergent was precipitated according to the Wessel and Fluegge method (Wessel and Flugge, 1984). SDS-PAGE was performed using the Laemmli method followed by immunoblotting as described previously (Laemmli, 1970).
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Acknowledgments |
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This work was supported by National Institutes of Health grants MH064794 (to B.G. Condie) and MH61934-04 (to E. Bieberich).
Submitted: 11 December 2002
Accepted: 2 June 2003
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References |
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Bhakar, A.L., L.-L. Tannis, C. Zeindler, M.P. Russo, C. Jobin, D.S. Park, S. MacPherson, and P.A. Barker. 2002. Constitutive nuclear factor-B activity is required for central neuron survival. J. Neurosci. 22:84668475.
Bieberich, E., S. MacKinnon, J. Silva, and R.K. Yu. 2001. Regulation of apoptosis during neuronal differentiation by ceramide and b-series complex gangliosides. J. Biol. Chem. 276:4439644404.
Blaschke, A.J., K. Staley, and J. Chun. 1996. Widespread programmed cell death in proliferative and post-mitotic regions of fetal cerebral cortex. Development. 122:11651174.
Camandola, S., and M.P. Mattson. 2000. Pro-apoptotic action of Par-4 involves inhibition of NF-B activity and suppression of bcl-2 expression. J. Neurosci. Res. 61:134139.[CrossRef][Medline]
Cecconi, F., G. Alvarez-Bolado, B.I. Meyer, K.A. Roth, and P. Gruss. 1998. Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell. 94:727737.[Medline]
Chan, W.Y., D.E. Lorke, S.C. Tiu, and D.T. Yew. 2002. Proliferation and apoptosis in the developing human neocortex. Anat. Rec. 267:261276.[CrossRef][Medline]
Cheema, S.K., S.K. Mishra, V.M. Rangnekar, A.M. Tari, R. Kumar, and G. Lopez-Berestein. 2003. PAR-4 transcriptionally regulates Bcl-2 through a WT1-binding site on the bcl-2 promoter. J. Biol. Chem. 278:1999520005. First published on March 17, 2003; 10.1074/jbc.M205865200.
Dasgupta, S., and E.L. Hogan. 2001. Chromatographic resolution and quantitative assay of CNS tissue sphingoids and sphingolipids. J. Lipid Res. 42:301308.
Diaz-Meco, M.T., M.M. Municio, S. Frutos, P. Sanchez, J. Lozano, L. Sanz, and J. Moscat. 1996. The product of par-4, a gene induced during apoptosis, interacts selectively with the atypical isoforms of protein kinase C. Cell. 86:777786.[Medline]
Fraichard, A., O. Chassande, G. Bilbaut, C. Dehay, P. Savatier, and J. Samarut. 1995. In vitro differentiation of embryonic stem cells into glial cells and functional neurons. J. Cell Sci. 108:31813188.
Grassme, H., A. Jekle, A. Riehle, H. Schwarz, J. Berger, K. Sandhoff, R. Kolesnick, and E. Gulbins. 2001. CD95 signaling via ceramide-rich membrane rafts. J. Biol. Chem. 276:2058920596.
Guo, Q., W. Fu, J. Xie, H. Luo, S.F. Sells, J.W. Geddes, V. Bondada, V. Rangnekar, and M.P. Mattson. 1998. Par-4 is a mediator of neuronal degeneration associated with the pathogenesis of Alzheimer disease. Nat. Med. 4:957962.[Medline]
Guo, Q., J. Xie, X. Chang, X. Zhang, and H. Du. 2001. Par-4 is a synaptic protein that regulates neurite outgrowth by altering calcium homeostasis and transcription factor AP-1 activation. Brain Res. 903:1325.[CrossRef][Medline]
Hakem, R., A. Hakem, G.S. Duncan, J.T. Henderson, M. Woo, M.S. Soengas, A. Elia, J.L. de la Pompa, D. Kagi, W. Khoo, et al. 1998. Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell. 94:339352.[Medline]
Hanada, K., T. Hara, M. Fukasawa, A. Yamaji, M. Umeda, and M. Nishijima. 1998. Mammalian cell mutants resistant to a sphingomyelin-directed cytolysin. Genetic and biochemical evidence for complex formation of the LCB1 protein with the LCB2 protein for serine palmitoyltransferase. J. Biol. Chem. 273:3378733794.
Hancock, C.R., J.P. Wetherington, N.A. Lambert, and B. Condie. 2000. Neuronal differentiation of cryopreserved neural progenitor cell derived form mouse embryonic stem cells. Biochem. Biophys. Res. Commun. 271:418421.[CrossRef][Medline]
Hatten, M.E. 1999. Central nervous system neuronal migration. Annu. Rev. Neurosci. 22:511539.[CrossRef][Medline]
Herget, T., C. Esdar, S.A. Oehrlein, M. Heinrich, S. Schutze, A. Maelicke, and G. van Echten-Deckert. 2000. Production of ceramides causes apoptosis during early neural differentiation in vitro. J. Biol. Chem. 275:3034430354.
Ji, L., G. Zhang, S. Uematsu, Y. Akahori, and Y. Hirabayashi. 1995. Induction of apoptotic DNA fragmentation and cell death by natural ceramide. FEBS Lett. 358:211214.[CrossRef][Medline]
Kuan, C.-Y., K.A. Roth, R.A. Flavell, and P. Rakic. 2000. Mechanisms of programmed cell death in the developing brain. Trends Neurosci. 23:291297.[CrossRef][Medline]
Kuida, K., T.F. Haydar, C.Y. Kuan, Y. Gu, C. Taya, H. Karasuyama, M.S.S. Su, P. Rakic, and R.A. Flavell. 1998. Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell. 94:325337.[Medline]
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage 4. Nature. 227:680685.[Medline]
Leblond, C.P., and M. El-Alfy. 1998. The eleven stages of the cell cycle, with emphasis on the changes in chromosomes and nucleoli during interphase and mitosis. Anat. Rec. 252:426443.[CrossRef][Medline]
Liu, D.X., and L.A. Greene. 2001. Neuronal apoptosis at the G1/S cell cycle checkpoint. Cell Tissue Res. 305:217228.[CrossRef][Medline]
Mattson, M.P. 2000. Apoptotic and anti-apoptotic synaptic signaling mechanisms. Brain Pathol. 10:300312.[Medline]
Mayer-Proschel, M., A.J. Kalyani, T. Mujitaba, and M.S. Rao. 1997. Isolation of lineage-restricted neuronal precursors from multipotent neuroepithelial stem cells. Neuron. 19:773785.[Medline]
Morcos, P.A. 2001. Achieving efficient delivery of morpholino oligos in cultured cells. Genesis. 30:94102.[CrossRef][Medline]
Movsesyan, V.A., A.G. Yakovlev, E.A. Dabaghyan, B.A. Stoica, and A.I. Faden. 2002. Ceramide induces neuronal apoptosis through the caspase-9/caspase-3 pathway. Biochem. Biophys. Res. Commun. 299:201207.[CrossRef][Medline]
Okabe, S., K. Forsberg-Nilsson, A.C. Spiro, M. Segal, and R.D. McKay. 1996. Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro. Mech. Dev. 59:89102.[CrossRef][Medline]
Orgogozo, V., F. Schweisguth, and Y. Bellaiche. 2002. Binary cell death decision regulated by unequal partitioning of Numb at mitosis. Development. 129:46774684.[Medline]
Pesce, M., and H.R. Scholer. 2001. Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells. 19:271278.
Shen, Q., W. Zhong, Y.N. Jan, and S. Temple. 2002. Asymmetric Numb distribution is critical for asymmetric cell division of mouse cerebral cortical stem cells and neuroblasts. Development. 129:48434853.[Medline]
Signorelli, P., and Y.A. Hannun. 2002. Analysis and quantitation of ceramide. Methods Enzymol. 345:275294.[Medline]
Sommer, L., and M. Rao. 2002. Neural stem cells and regulation of cell number. Prog. Neurobiol. 66:118.[CrossRef][Medline]
Toman, R.E., V. Movsesyan, S.K. Murthy, S. Milstien, S. Spiegel, and A.I. Faden. 2002. Ceramide induced cell death in primary neuronal cultures: upregulation of ceramide levels during neuronal apoptosis. J. Neurosci. Res. 68:323330.[CrossRef][Medline]
Wang, C.-S., and R.L. Smith. 1975. Lowry determination of protein in the presence of Triton X-100. Anal. Biochem. 63:414417.[Medline]
Wessel, D., and U.I. Flugge. 1984. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138:141143.[Medline]
Wu, Y.Y., T. Mujtaba, S.S.W. Han, I. Fischer, and M.S. Rao. 2002. Isolation of a glial-restricted tripotential cell line from embryonal spinal cord cultures. Glia. 38:6579.[CrossRef][Medline]
Xie, J., X. Chang, X. Zhang, and Q. Guo. 2001. Aberrant induction of Par-4 is involved in apoptosis of hippocampal neurons in presenilin-1 M146V mutant knock-in mice. Brain Res. 915:110.[CrossRef][Medline]
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