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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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Apoptosis of differentiating ES-J1 ES cells at the NP2 stage of differentiation. Multiple, indirect immunofluorescence staining of differentiating ES-J1 cells 48 h on replating of EB8 cells (NP2 stage). (A) Nestin, (Cy3, red); PCNA, (Cy5, far-red, pseudo-colored as pink); TUNEL, FITC (green); Hoechst (blue). White color shows PCNA- and TUNEL-stained (apoptotic) cells. Pink staining shows cells that are PCNA-positive, but not apoptotic. (B) Cells at the NP2 stage were pulse labeled for 3 h with BrdU, followed by fixation and immunostaining for BrdU (green), activated caspase 3 (Cy5, pseudo-colored as pink), and Hoechst (blue) 5 h after labeling. White staining shows TUNEL- and active caspase 3–stained cells. Pink staining shows only activated caspase 3–positive cells.
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fig7: Apoptosis of differentiating ES-J1 ES cells at the NP2 stage of differentiation. Multiple, indirect immunofluorescence staining of differentiating ES-J1 cells 48 h on replating of EB8 cells (NP2 stage). (A) Nestin, (Cy3, red); PCNA, (Cy5, far-red, pseudo-colored as pink); TUNEL, FITC (green); Hoechst (blue). White color shows PCNA- and TUNEL-stained (apoptotic) cells. Pink staining shows cells that are PCNA-positive, but not apoptotic. (B) Cells at the NP2 stage were pulse labeled for 3 h with BrdU, followed by fixation and immunostaining for BrdU (green), activated caspase 3 (Cy5, pseudo-colored as pink), and Hoechst (blue) 5 h after labeling. White staining shows TUNEL- and active caspase 3–stained cells. Pink staining shows only activated caspase 3–positive cells.

Mentions: 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).


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)

Apoptosis of differentiating ES-J1 ES cells at the NP2 stage of differentiation. Multiple, indirect immunofluorescence staining of differentiating ES-J1 cells 48 h on replating of EB8 cells (NP2 stage). (A) Nestin, (Cy3, red); PCNA, (Cy5, far-red, pseudo-colored as pink); TUNEL, FITC (green); Hoechst (blue). White color shows PCNA- and TUNEL-stained (apoptotic) cells. Pink staining shows cells that are PCNA-positive, but not apoptotic. (B) Cells at the NP2 stage were pulse labeled for 3 h with BrdU, followed by fixation and immunostaining for BrdU (green), activated caspase 3 (Cy5, pseudo-colored as pink), and Hoechst (blue) 5 h after labeling. White staining shows TUNEL- and active caspase 3–stained cells. Pink staining shows only activated caspase 3–positive cells.
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fig7: Apoptosis of differentiating ES-J1 ES cells at the NP2 stage of differentiation. Multiple, indirect immunofluorescence staining of differentiating ES-J1 cells 48 h on replating of EB8 cells (NP2 stage). (A) Nestin, (Cy3, red); PCNA, (Cy5, far-red, pseudo-colored as pink); TUNEL, FITC (green); Hoechst (blue). White color shows PCNA- and TUNEL-stained (apoptotic) cells. Pink staining shows cells that are PCNA-positive, but not apoptotic. (B) Cells at the NP2 stage were pulse labeled for 3 h with BrdU, followed by fixation and immunostaining for BrdU (green), activated caspase 3 (Cy5, pseudo-colored as pink), and Hoechst (blue) 5 h after labeling. White staining shows TUNEL- and active caspase 3–stained cells. Pink staining shows only activated caspase 3–positive cells.
Mentions: 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).

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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