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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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Distribution of ceramide and PAR-4 during cell division of ES-J1 ES cells at the NP2 stage of differentiation and caspase 3 activation. Multiple, indirect immunofluorescence staining of differentiating ES-J1 cells 48 h after replating of EB8 cells (NP2 stage). Ceramide, FITC (green); PAR-4, (Alexa® 546, red); Hoechst (blue). A, mitotic cell in anaphase; B, mitotic cell in telophase; C, nonapoptotic cell; D–D′′, apoptotic cell. Leftmost picture (D) shows overlay of ceramide (D′), PAR-4 (D′′), and Hoechst staining; E, overlays of TUNEL (green) and PAR-4 (red) staining or E′, TUNEL (green) and activated caspase 3 (Cy5, pseudo-colored as pink) staining.
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fig8: Distribution of ceramide and PAR-4 during cell division of ES-J1 ES cells at the NP2 stage of differentiation and caspase 3 activation. Multiple, indirect immunofluorescence staining of differentiating ES-J1 cells 48 h after replating of EB8 cells (NP2 stage). Ceramide, FITC (green); PAR-4, (Alexa® 546, red); Hoechst (blue). A, mitotic cell in anaphase; B, mitotic cell in telophase; C, nonapoptotic cell; D–D′′, apoptotic cell. Leftmost picture (D) shows overlay of ceramide (D′), PAR-4 (D′′), and Hoechst staining; E, overlays of TUNEL (green) and PAR-4 (red) staining or E′, TUNEL (green) and activated caspase 3 (Cy5, pseudo-colored as pink) staining.

Mentions: The results from ceramide and PAR-4 double-immunostaining experiments suggested that either ceramide or PAR-4 was sequestered to only one daughter cell during cell division. We identified mitotic cells in anaphase (mitosis stage VII) and telophase (mitosis stage VIII) by the polarized orientation of nuclei on fixation and staining with Hoechst dye, a useful marker for the identification of different mitotic stages (Leblond and El-Alfy, 1998). 10 out of 5,000 cells were found to be in mitosis stage VII or VIII. Ceramide (Fig. 8, green) was homogenously distributed to the two daughter cells, whereas PAR-4 (Fig. 8, red) was sequestered to only one daughter cell (Fig. 8 B). The daughter cells with elevated PAR-4 appeared apoptotic, as PAR-4 colocalized with ceramide in membrane blebs that are typical for apoptosis (Fig. 8, D–D′′). Ceramide-induced apoptosis in PAR-4–positive cells was executed by the caspase 9-to-caspase 3 cascade as shown by coimmunostaining of elevated PAR-4 and cleaved caspase 3 in TUNEL–positive cells (Fig. 8, E and E′).


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)

Distribution of ceramide and PAR-4 during cell division of ES-J1 ES cells at the NP2 stage of differentiation and caspase 3 activation. Multiple, indirect immunofluorescence staining of differentiating ES-J1 cells 48 h after replating of EB8 cells (NP2 stage). Ceramide, FITC (green); PAR-4, (Alexa® 546, red); Hoechst (blue). A, mitotic cell in anaphase; B, mitotic cell in telophase; C, nonapoptotic cell; D–D′′, apoptotic cell. Leftmost picture (D) shows overlay of ceramide (D′), PAR-4 (D′′), and Hoechst staining; E, overlays of TUNEL (green) and PAR-4 (red) staining or E′, TUNEL (green) and activated caspase 3 (Cy5, pseudo-colored as pink) staining.
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Related In: Results  -  Collection

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fig8: Distribution of ceramide and PAR-4 during cell division of ES-J1 ES cells at the NP2 stage of differentiation and caspase 3 activation. Multiple, indirect immunofluorescence staining of differentiating ES-J1 cells 48 h after replating of EB8 cells (NP2 stage). Ceramide, FITC (green); PAR-4, (Alexa® 546, red); Hoechst (blue). A, mitotic cell in anaphase; B, mitotic cell in telophase; C, nonapoptotic cell; D–D′′, apoptotic cell. Leftmost picture (D) shows overlay of ceramide (D′), PAR-4 (D′′), and Hoechst staining; E, overlays of TUNEL (green) and PAR-4 (red) staining or E′, TUNEL (green) and activated caspase 3 (Cy5, pseudo-colored as pink) staining.
Mentions: The results from ceramide and PAR-4 double-immunostaining experiments suggested that either ceramide or PAR-4 was sequestered to only one daughter cell during cell division. We identified mitotic cells in anaphase (mitosis stage VII) and telophase (mitosis stage VIII) by the polarized orientation of nuclei on fixation and staining with Hoechst dye, a useful marker for the identification of different mitotic stages (Leblond and El-Alfy, 1998). 10 out of 5,000 cells were found to be in mitosis stage VII or VIII. Ceramide (Fig. 8, green) was homogenously distributed to the two daughter cells, whereas PAR-4 (Fig. 8, red) was sequestered to only one daughter cell (Fig. 8 B). The daughter cells with elevated PAR-4 appeared apoptotic, as PAR-4 colocalized with ceramide in membrane blebs that are typical for apoptosis (Fig. 8, D–D′′). Ceramide-induced apoptosis in PAR-4–positive cells was executed by the caspase 9-to-caspase 3 cascade as shown by coimmunostaining of elevated PAR-4 and cleaved caspase 3 in TUNEL–positive cells (Fig. 8, E and E′).

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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