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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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Alteration of neural stem cell apoptosis by inhibition of ceramide biosynthesis or incubation with ceramide analogs. (A–C) Differentiating ES cells at the NP2 stage were immunostained for PAR-4 (red, left) and nestin (red, right), apoptotic cells were TUNEL stained (green), and nuclei were counterstained with Hoechst dye (blue). A, control cells without FB1 or the novel ceramide analogue S18; B, cells incubated for 48 h with 25 μM FB1; C, cells incubated for 48 h with FB1 (ceramide depletion) followed by overnight treatment with 80 μM S18. Arrows indicate apoptotic cells.
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fig4: Alteration of neural stem cell apoptosis by inhibition of ceramide biosynthesis or incubation with ceramide analogs. (A–C) Differentiating ES cells at the NP2 stage were immunostained for PAR-4 (red, left) and nestin (red, right), apoptotic cells were TUNEL stained (green), and nuclei were counterstained with Hoechst dye (blue). A, control cells without FB1 or the novel ceramide analogue S18; B, cells incubated for 48 h with 25 μM FB1; C, cells incubated for 48 h with FB1 (ceramide depletion) followed by overnight treatment with 80 μM S18. Arrows indicate apoptotic cells.

Mentions: We determined the degree of apoptosis in differentiating ES cultures because we have previously proposed that ceramide elevation induces apoptosis in NP cells (Bieberich et al., 2001). As shown in Fig. 3 B, the degree of apoptosis was quantified by counting TUNEL-stained cells. Apoptosis was elevated at the EB8 stage (20 ± 5%) and was highest at the NP2 stage (35 ± 5%). The fraction of apoptotic cells rapidly decreased on induction of neural differentiation, and was already <20% at D1. Differentiated neurons at D4 did not show a significant degree of apoptosis (<10 ± 5%). These results show that the peak time of apoptosis coincided with the peak elevation of endogenous ceramide. Consistent with this, inhibition of ceramide biosynthesis by incubation with 25 μM of the ceramide synthase inhibitor fumonisin B1 (FB1) or 50 nM of the serine palmitoyltransferase (SPT) inhibitor myriocin for 48 h reduced the degree of apoptosis at the EB8 and NP2 stage by ∼50%. Fig. 4 (right) shows that at the NP2 stage, apoptosis proceeded in differentiating ES cells that did not stain for nestin (Fig. 4 A). In these cells, FB1 significantly reduced the degree of apoptosis (Fig. 4 B). Apoptosis was restored by the addition of 30 μM N-acetyl sphingosine (C2-ceramide; not depicted), 80 μM of the novel ceramide analogue N-oleoyl serinol (Fig. 4 C, S18; for review see Bieberich et al., 2001), or 1 μM of natural ceramide (not depicted) that was extracted from differentiating ES cells. Together, these results showed that elevation of endogenous ceramide was a prerequisite for the induction of apoptosis in differentiating ES cells at the NP2 stage.


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)

Alteration of neural stem cell apoptosis by inhibition of ceramide biosynthesis or incubation with ceramide analogs. (A–C) Differentiating ES cells at the NP2 stage were immunostained for PAR-4 (red, left) and nestin (red, right), apoptotic cells were TUNEL stained (green), and nuclei were counterstained with Hoechst dye (blue). A, control cells without FB1 or the novel ceramide analogue S18; B, cells incubated for 48 h with 25 μM FB1; C, cells incubated for 48 h with FB1 (ceramide depletion) followed by overnight treatment with 80 μM S18. Arrows indicate apoptotic cells.
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Related In: Results  -  Collection

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fig4: Alteration of neural stem cell apoptosis by inhibition of ceramide biosynthesis or incubation with ceramide analogs. (A–C) Differentiating ES cells at the NP2 stage were immunostained for PAR-4 (red, left) and nestin (red, right), apoptotic cells were TUNEL stained (green), and nuclei were counterstained with Hoechst dye (blue). A, control cells without FB1 or the novel ceramide analogue S18; B, cells incubated for 48 h with 25 μM FB1; C, cells incubated for 48 h with FB1 (ceramide depletion) followed by overnight treatment with 80 μM S18. Arrows indicate apoptotic cells.
Mentions: We determined the degree of apoptosis in differentiating ES cultures because we have previously proposed that ceramide elevation induces apoptosis in NP cells (Bieberich et al., 2001). As shown in Fig. 3 B, the degree of apoptosis was quantified by counting TUNEL-stained cells. Apoptosis was elevated at the EB8 stage (20 ± 5%) and was highest at the NP2 stage (35 ± 5%). The fraction of apoptotic cells rapidly decreased on induction of neural differentiation, and was already <20% at D1. Differentiated neurons at D4 did not show a significant degree of apoptosis (<10 ± 5%). These results show that the peak time of apoptosis coincided with the peak elevation of endogenous ceramide. Consistent with this, inhibition of ceramide biosynthesis by incubation with 25 μM of the ceramide synthase inhibitor fumonisin B1 (FB1) or 50 nM of the serine palmitoyltransferase (SPT) inhibitor myriocin for 48 h reduced the degree of apoptosis at the EB8 and NP2 stage by ∼50%. Fig. 4 (right) shows that at the NP2 stage, apoptosis proceeded in differentiating ES cells that did not stain for nestin (Fig. 4 A). In these cells, FB1 significantly reduced the degree of apoptosis (Fig. 4 B). Apoptosis was restored by the addition of 30 μM N-acetyl sphingosine (C2-ceramide; not depicted), 80 μM of the novel ceramide analogue N-oleoyl serinol (Fig. 4 C, S18; for review see Bieberich et al., 2001), or 1 μM of natural ceramide (not depicted) that was extracted from differentiating ES cells. Together, these results showed that elevation of endogenous ceramide was a prerequisite for the induction of apoptosis in differentiating ES cells at the NP2 stage.

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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