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Regulation of cell death in mitotic neural progenitor cells by asymmetric distribution of prostate apoptosis response 4 (PAR-4) and simultaneous elevation of endogenous ceramide.

Bieberich E, MacKinnon S, Silva J, Noggle S, Condie BG - J. Cell Biol. (2003)

Bottom Line: Morpholino oligonucleotide-mediated antisense knockdown of PAR-4 or inhibition of ceramide biosynthesis reduced stem cell apoptosis, whereas PAR-4 overexpression and treatment with ceramide analogs elevated apoptosis.In mitotic cells, asymmetric distribution of PAR-4 and nestin resulted in one nestin(-)/PAR-4(+) daughter cell, in which ceramide elevation induced apoptosis.Asymmetric distribution of PAR-4 and simultaneous elevation of endogenous ceramide provides a possible mechanism underlying asymmetric differentiation and apoptosis of neuronal stem cells in the developing brain.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th Street, Room CB-2803, Augusta, GA 30912, USA. ebieberich@mail.mcg.edu

ABSTRACT
Cell death and survival of neural progenitor (NP) cells are determined by signals that are largely unknown. We have analyzed pro-apoptotic signaling in individual NP cells that have been derived from mouse embryonic stem cells. NP formation was concomitant with elevated apoptosis and increased expression of ceramide and prostate apoptosis response 4 (PAR-4). Morpholino oligonucleotide-mediated antisense knockdown of PAR-4 or inhibition of ceramide biosynthesis reduced stem cell apoptosis, whereas PAR-4 overexpression and treatment with ceramide analogs elevated apoptosis. Apoptotic cells also stained for proliferating cell nuclear antigen (a nuclear mitosis marker protein), but not for nestin (a marker for NP cells). In mitotic cells, asymmetric distribution of PAR-4 and nestin resulted in one nestin(-)/PAR-4(+) daughter cell, in which ceramide elevation induced apoptosis. The other cell was nestin(+), but PAR-4(-), and was not apoptotic. Asymmetric distribution of PAR-4 and simultaneous elevation of endogenous ceramide provides a possible mechanism underlying asymmetric differentiation and apoptosis of neuronal stem cells in the developing brain.

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Ceramide content and apoptosis during in vitro neuronal differentiation of ES cells. (A) Neutral lipids were purified from differentiating ES cells and a lipid amount corresponding to 750 μg of cellular protein/lane separated by HPTLC in the running solvent CH3Cl/HOAc (9:1, vol/vol). Lipids were stained with the cupric acetate reagent. Ceramide (open bars) was quantified by densitometric analysis and comparison with known amounts of standard lipid (N-oleoyl sphingosine). Lane 1, ceramide standard from bovine brain; lane 2, fibroblast-freed ES cells, after 4 d in culture; lane 3, EBs at the EB4 stage; lane 4, EBs at the EB8 stage; lane 5, NPs at the NP2 stage; lanes 6–8, three terminal differentiation stages, 24 (D1 stage), 48 (D2 stage), and 96 h (D4 stage) on cultivation of NP cells in serum-containing medium; lanes 9–11, N-oleoyl sphingosine, 250, 500, and 1,000 ng, respectively. (B) Lipids were extracted from differentiated ES cells and the amount of ceramide quantified using the DAG kinase assay. Apoptosis (solid bars) was determined by TUNEL staining. For HPTLC, DAG kinase, and TUNEL analyses, experiments were performed with five independent ES cultures. The bars show the standard mean and deviation of percentage of TUNEL-positive cells that were counted in five areas of 200 cells in each experiment.
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fig3: Ceramide content and apoptosis during in vitro neuronal differentiation of ES cells. (A) Neutral lipids were purified from differentiating ES cells and a lipid amount corresponding to 750 μg of cellular protein/lane separated by HPTLC in the running solvent CH3Cl/HOAc (9:1, vol/vol). Lipids were stained with the cupric acetate reagent. Ceramide (open bars) was quantified by densitometric analysis and comparison with known amounts of standard lipid (N-oleoyl sphingosine). Lane 1, ceramide standard from bovine brain; lane 2, fibroblast-freed ES cells, after 4 d in culture; lane 3, EBs at the EB4 stage; lane 4, EBs at the EB8 stage; lane 5, NPs at the NP2 stage; lanes 6–8, three terminal differentiation stages, 24 (D1 stage), 48 (D2 stage), and 96 h (D4 stage) on cultivation of NP cells in serum-containing medium; lanes 9–11, N-oleoyl sphingosine, 250, 500, and 1,000 ng, respectively. (B) Lipids were extracted from differentiated ES cells and the amount of ceramide quantified using the DAG kinase assay. Apoptosis (solid bars) was determined by TUNEL staining. For HPTLC, DAG kinase, and TUNEL analyses, experiments were performed with five independent ES cultures. The bars show the standard mean and deviation of percentage of TUNEL-positive cells that were counted in five areas of 200 cells in each experiment.

Mentions: To measure ceramide levels during ES cell differentiation, 50–100 mg of cells were harvested at different time points during differentiation. Sphingolipids were isolated using organic solvent extraction. As shown in Fig. 3 (A and B), quantitative HPTLC and DAG kinase assay revealed that fibroblast-free, undifferentiated ES cells (Fig. 3 A, lane 2) contained <0.2 ± 0.1 μg ceramide/mg cell protein. After 4 d of EB formation (EB4 stage), ceramide had increased to 0.4 ± 0.1 μg ceramide/mg cell protein (Fig. 3 A, lane 3). Endogenous ceramide was further elevated to 1.0 ± 0.2 μg/mg cell protein by the EB8 stage of differentiation (Fig. 3 A, lane 4). The increased ceramide concentration was maintained through the NP2 stage of differentiation (Fig. 3 A, lane 5). In the D1 stage of differentiation, the ceramide concentration was found to be reduced by 70% (0.3 ± 0.1 μg ceramide/mg cell protein) and did not change significantly after 4 d of differentiation (Fig. 3 A; D1, D2, and D4, lanes 6, 7, and 8, respectively). Together, these results indicate that the peak elevation of ceramide occurs during the initial formation of NP cells on serum deprivation of EBs and during NP expansion in the presence of FGF-2.


Regulation of cell death in mitotic neural progenitor cells by asymmetric distribution of prostate apoptosis response 4 (PAR-4) and simultaneous elevation of endogenous ceramide.

Bieberich E, MacKinnon S, Silva J, Noggle S, Condie BG - J. Cell Biol. (2003)

Ceramide content and apoptosis during in vitro neuronal differentiation of ES cells. (A) Neutral lipids were purified from differentiating ES cells and a lipid amount corresponding to 750 μg of cellular protein/lane separated by HPTLC in the running solvent CH3Cl/HOAc (9:1, vol/vol). Lipids were stained with the cupric acetate reagent. Ceramide (open bars) was quantified by densitometric analysis and comparison with known amounts of standard lipid (N-oleoyl sphingosine). Lane 1, ceramide standard from bovine brain; lane 2, fibroblast-freed ES cells, after 4 d in culture; lane 3, EBs at the EB4 stage; lane 4, EBs at the EB8 stage; lane 5, NPs at the NP2 stage; lanes 6–8, three terminal differentiation stages, 24 (D1 stage), 48 (D2 stage), and 96 h (D4 stage) on cultivation of NP cells in serum-containing medium; lanes 9–11, N-oleoyl sphingosine, 250, 500, and 1,000 ng, respectively. (B) Lipids were extracted from differentiated ES cells and the amount of ceramide quantified using the DAG kinase assay. Apoptosis (solid bars) was determined by TUNEL staining. For HPTLC, DAG kinase, and TUNEL analyses, experiments were performed with five independent ES cultures. The bars show the standard mean and deviation of percentage of TUNEL-positive cells that were counted in five areas of 200 cells in each experiment.
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Related In: Results  -  Collection

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fig3: Ceramide content and apoptosis during in vitro neuronal differentiation of ES cells. (A) Neutral lipids were purified from differentiating ES cells and a lipid amount corresponding to 750 μg of cellular protein/lane separated by HPTLC in the running solvent CH3Cl/HOAc (9:1, vol/vol). Lipids were stained with the cupric acetate reagent. Ceramide (open bars) was quantified by densitometric analysis and comparison with known amounts of standard lipid (N-oleoyl sphingosine). Lane 1, ceramide standard from bovine brain; lane 2, fibroblast-freed ES cells, after 4 d in culture; lane 3, EBs at the EB4 stage; lane 4, EBs at the EB8 stage; lane 5, NPs at the NP2 stage; lanes 6–8, three terminal differentiation stages, 24 (D1 stage), 48 (D2 stage), and 96 h (D4 stage) on cultivation of NP cells in serum-containing medium; lanes 9–11, N-oleoyl sphingosine, 250, 500, and 1,000 ng, respectively. (B) Lipids were extracted from differentiated ES cells and the amount of ceramide quantified using the DAG kinase assay. Apoptosis (solid bars) was determined by TUNEL staining. For HPTLC, DAG kinase, and TUNEL analyses, experiments were performed with five independent ES cultures. The bars show the standard mean and deviation of percentage of TUNEL-positive cells that were counted in five areas of 200 cells in each experiment.
Mentions: To measure ceramide levels during ES cell differentiation, 50–100 mg of cells were harvested at different time points during differentiation. Sphingolipids were isolated using organic solvent extraction. As shown in Fig. 3 (A and B), quantitative HPTLC and DAG kinase assay revealed that fibroblast-free, undifferentiated ES cells (Fig. 3 A, lane 2) contained <0.2 ± 0.1 μg ceramide/mg cell protein. After 4 d of EB formation (EB4 stage), ceramide had increased to 0.4 ± 0.1 μg ceramide/mg cell protein (Fig. 3 A, lane 3). Endogenous ceramide was further elevated to 1.0 ± 0.2 μg/mg cell protein by the EB8 stage of differentiation (Fig. 3 A, lane 4). The increased ceramide concentration was maintained through the NP2 stage of differentiation (Fig. 3 A, lane 5). In the D1 stage of differentiation, the ceramide concentration was found to be reduced by 70% (0.3 ± 0.1 μg ceramide/mg cell protein) and did not change significantly after 4 d of differentiation (Fig. 3 A; D1, D2, and D4, lanes 6, 7, and 8, respectively). Together, these results indicate that the peak elevation of ceramide occurs during the initial formation of NP cells on serum deprivation of EBs and during NP expansion in the presence of FGF-2.

Bottom Line: Morpholino oligonucleotide-mediated antisense knockdown of PAR-4 or inhibition of ceramide biosynthesis reduced stem cell apoptosis, whereas PAR-4 overexpression and treatment with ceramide analogs elevated apoptosis.In mitotic cells, asymmetric distribution of PAR-4 and nestin resulted in one nestin(-)/PAR-4(+) daughter cell, in which ceramide elevation induced apoptosis.Asymmetric distribution of PAR-4 and simultaneous elevation of endogenous ceramide provides a possible mechanism underlying asymmetric differentiation and apoptosis of neuronal stem cells in the developing brain.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th Street, Room CB-2803, Augusta, GA 30912, USA. ebieberich@mail.mcg.edu

ABSTRACT
Cell death and survival of neural progenitor (NP) cells are determined by signals that are largely unknown. We have analyzed pro-apoptotic signaling in individual NP cells that have been derived from mouse embryonic stem cells. NP formation was concomitant with elevated apoptosis and increased expression of ceramide and prostate apoptosis response 4 (PAR-4). Morpholino oligonucleotide-mediated antisense knockdown of PAR-4 or inhibition of ceramide biosynthesis reduced stem cell apoptosis, whereas PAR-4 overexpression and treatment with ceramide analogs elevated apoptosis. Apoptotic cells also stained for proliferating cell nuclear antigen (a nuclear mitosis marker protein), but not for nestin (a marker for NP cells). In mitotic cells, asymmetric distribution of PAR-4 and nestin resulted in one nestin(-)/PAR-4(+) daughter cell, in which ceramide elevation induced apoptosis. The other cell was nestin(+), but PAR-4(-), and was not apoptotic. Asymmetric distribution of PAR-4 and simultaneous elevation of endogenous ceramide provides a possible mechanism underlying asymmetric differentiation and apoptosis of neuronal stem cells in the developing brain.

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