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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 PAR-4 and nestin during cell division of ES-J1 ES cells at NP2 stage of differentiation. Multiple, indirect immunofluorescence staining of differentiating ES-J1 cells 48 h on replating of EB8 cells (NP2 stage). PAR-4, (Alexa® 546, red); nestin, (Alexa® 488, green); Hoechst (blue). (A) Mitotic cell in anaphase/telophase, overlay of PAR-4, nestin, and Hoechst staining. Note the strict asymmetric distribution of PAR-4 and nestin. (B) Cluster of neuroprogenitor cells, overlay of PAR-4, nestin, and Hoechst staining. Arrowheads indicate two apoptotic PAR-4–positive cells that did not show any expression of nestin.
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fig9: Distribution of PAR-4 and nestin during cell division of ES-J1 ES cells at NP2 stage of differentiation. Multiple, indirect immunofluorescence staining of differentiating ES-J1 cells 48 h on replating of EB8 cells (NP2 stage). PAR-4, (Alexa® 546, red); nestin, (Alexa® 488, green); Hoechst (blue). (A) Mitotic cell in anaphase/telophase, overlay of PAR-4, nestin, and Hoechst staining. Note the strict asymmetric distribution of PAR-4 and nestin. (B) Cluster of neuroprogenitor cells, overlay of PAR-4, nestin, and Hoechst staining. Arrowheads indicate two apoptotic PAR-4–positive cells that did not show any expression of nestin.

Mentions: The lack of nestin expression in PAR-4–positive cells suggested that nestin and PAR-4 were sequestered asymmetrically to the daughters of dividing NP cells. Fig. 9 shows that immunostaining for nestin (green) and PAR-4 (red) was indeed asymmetrically distributed in mitosis stage VIII cells (Fig. 9 A). The PAR-4–positive (but nestin-negative) cells underwent apoptosis (top right), whereas the nestin-positive cells were nonapoptotic and expressed either none or significantly less PAR-4 than nestin-negative cells. The absence of nestin staining in apoptotic cells (Fig. 9 B, arrows) is consistent with the results shown in Table II and Fig. 4, Fig. 5, and Fig. 7. These data are also consistent with the low number of cells (6%) that costained for PAR-4 and nestin (Table II). In cases where residual PAR-4 was present in nestin-positive daughter cells, the PAR-4 and nestin were clearly separated (Fig. 9 B). Together, these observations suggest that PAR-4 is partitioned specifically into nestin-negative daughter cells.


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 PAR-4 and nestin during cell division of ES-J1 ES cells at NP2 stage of differentiation. Multiple, indirect immunofluorescence staining of differentiating ES-J1 cells 48 h on replating of EB8 cells (NP2 stage). PAR-4, (Alexa® 546, red); nestin, (Alexa® 488, green); Hoechst (blue). (A) Mitotic cell in anaphase/telophase, overlay of PAR-4, nestin, and Hoechst staining. Note the strict asymmetric distribution of PAR-4 and nestin. (B) Cluster of neuroprogenitor cells, overlay of PAR-4, nestin, and Hoechst staining. Arrowheads indicate two apoptotic PAR-4–positive cells that did not show any expression of nestin.
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Related In: Results  -  Collection

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fig9: Distribution of PAR-4 and nestin during cell division of ES-J1 ES cells at NP2 stage of differentiation. Multiple, indirect immunofluorescence staining of differentiating ES-J1 cells 48 h on replating of EB8 cells (NP2 stage). PAR-4, (Alexa® 546, red); nestin, (Alexa® 488, green); Hoechst (blue). (A) Mitotic cell in anaphase/telophase, overlay of PAR-4, nestin, and Hoechst staining. Note the strict asymmetric distribution of PAR-4 and nestin. (B) Cluster of neuroprogenitor cells, overlay of PAR-4, nestin, and Hoechst staining. Arrowheads indicate two apoptotic PAR-4–positive cells that did not show any expression of nestin.
Mentions: The lack of nestin expression in PAR-4–positive cells suggested that nestin and PAR-4 were sequestered asymmetrically to the daughters of dividing NP cells. Fig. 9 shows that immunostaining for nestin (green) and PAR-4 (red) was indeed asymmetrically distributed in mitosis stage VIII cells (Fig. 9 A). The PAR-4–positive (but nestin-negative) cells underwent apoptosis (top right), whereas the nestin-positive cells were nonapoptotic and expressed either none or significantly less PAR-4 than nestin-negative cells. The absence of nestin staining in apoptotic cells (Fig. 9 B, arrows) is consistent with the results shown in Table II and Fig. 4, Fig. 5, and Fig. 7. These data are also consistent with the low number of cells (6%) that costained for PAR-4 and nestin (Table II). In cases where residual PAR-4 was present in nestin-positive daughter cells, the PAR-4 and nestin were clearly separated (Fig. 9 B). Together, these observations suggest that PAR-4 is partitioned specifically into nestin-negative daughter cells.

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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