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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 antisense knockdown or overexpression of PAR-4. (A) Differentiating ES cells at the NP1 stage were transfected with a morpholino phosphorodiamidate antisense oligonucleotide against PAR-4, and 48 h later (NP3 stage), were incubated overnight with 80 μM S18. The figure shows PAR-4 (red), nestin (green), and TUNEL (blue) staining of cells transfected with standard control antisense oligonucleotide (left), and cells transfected with PAR-4–specific antisense oligonucleotide followed by incubation with S18 (right). (B) Staining of PAR-4 on immunoblots of protein from differentiating ES cells (NP3 stage) that were transfected with or without PAR-4–specific antisense oligonucleotide or PAR-4-RFP, respectively. Lane 1, untransfected (UT) NP cells; lane 2, NP cells transfected with standard control antisense oligonucleotide (Con); lane 3, NP cells transfected with PAR-4–specific antisense oligonucleotide (Anti); lane 4, NP cells transfected with PAR-4-RFP. (C) In differentiating ES cells at the EB8 stage, inhibition of ceramide biosynthesis was initiated by incubation with 50 nM myriocin, and was then maintained throughout the subsequent differentiation stages. After NP expansion (NP1 stage), cells were transfected with PAR-4-RFP, and 48 h later (NP3 stage), were incubated overnight with 80 μM S18 (right). The figure shows phase contrast and overlay with RFP (red) fluorescence. Arrow in A indicates apoptotic cells.
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fig5: Alteration of neural stem cell apoptosis by antisense knockdown or overexpression of PAR-4. (A) Differentiating ES cells at the NP1 stage were transfected with a morpholino phosphorodiamidate antisense oligonucleotide against PAR-4, and 48 h later (NP3 stage), were incubated overnight with 80 μM S18. The figure shows PAR-4 (red), nestin (green), and TUNEL (blue) staining of cells transfected with standard control antisense oligonucleotide (left), and cells transfected with PAR-4–specific antisense oligonucleotide followed by incubation with S18 (right). (B) Staining of PAR-4 on immunoblots of protein from differentiating ES cells (NP3 stage) that were transfected with or without PAR-4–specific antisense oligonucleotide or PAR-4-RFP, respectively. Lane 1, untransfected (UT) NP cells; lane 2, NP cells transfected with standard control antisense oligonucleotide (Con); lane 3, NP cells transfected with PAR-4–specific antisense oligonucleotide (Anti); lane 4, NP cells transfected with PAR-4-RFP. (C) In differentiating ES cells at the EB8 stage, inhibition of ceramide biosynthesis was initiated by incubation with 50 nM myriocin, and was then maintained throughout the subsequent differentiation stages. After NP expansion (NP1 stage), cells were transfected with PAR-4-RFP, and 48 h later (NP3 stage), were incubated overnight with 80 μM S18 (right). The figure shows phase contrast and overlay with RFP (red) fluorescence. Arrow in A indicates apoptotic cells.

Mentions: To suppress PAR-4 expression, ES cells at the NP1 stage were transfected with a PAR-4–specific morpholino phosphorodiamidate antisense oligonucleotide (morpholino) that was designed on the basis of a previously published antisense oligonucleotide sequence (Guo et al., 1998, 2001). Morpholinos have been shown to avoid many of the pitfalls associated with conventional antisense oligonucleotides, and have been successfully used for transfection of embryos and cultured cells (Morcos, 2001). Fig. 5 A (left) shows NP cells 48 h after transfection with a standard morpholino provided as negative control. The number of TUNEL-stained cells in the negative control was equivalent to that found with untransfected cells (35%, see Fig. 3 B), indicating that the degree of apoptosis was not affected due to any unspecific effect of the morpholino. This result was consistent with the observation that the expression level of PAR-4 in untransfected and control morpholino-transfected NP cells was the same (Fig. 5 B, lane 1 and lane 2). Incubation of control morpholino-transfected NP cells with 80 μM S18 increased the number of apoptotic cells to that found with S18-treated, untransfected cells (>80%). However, transfection of NP cells with a PAR-4–specific antisense morpholino reduced S18-induced apoptosis to a level <30% (Fig. 5 A, right). Consistently, transfection with the PAR-4 antisense morpholino suppressed the expression level of PAR-4 to ∼25% of that detected in untransfected cells (Fig. 5 B, lane 3).


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 antisense knockdown or overexpression of PAR-4. (A) Differentiating ES cells at the NP1 stage were transfected with a morpholino phosphorodiamidate antisense oligonucleotide against PAR-4, and 48 h later (NP3 stage), were incubated overnight with 80 μM S18. The figure shows PAR-4 (red), nestin (green), and TUNEL (blue) staining of cells transfected with standard control antisense oligonucleotide (left), and cells transfected with PAR-4–specific antisense oligonucleotide followed by incubation with S18 (right). (B) Staining of PAR-4 on immunoblots of protein from differentiating ES cells (NP3 stage) that were transfected with or without PAR-4–specific antisense oligonucleotide or PAR-4-RFP, respectively. Lane 1, untransfected (UT) NP cells; lane 2, NP cells transfected with standard control antisense oligonucleotide (Con); lane 3, NP cells transfected with PAR-4–specific antisense oligonucleotide (Anti); lane 4, NP cells transfected with PAR-4-RFP. (C) In differentiating ES cells at the EB8 stage, inhibition of ceramide biosynthesis was initiated by incubation with 50 nM myriocin, and was then maintained throughout the subsequent differentiation stages. After NP expansion (NP1 stage), cells were transfected with PAR-4-RFP, and 48 h later (NP3 stage), were incubated overnight with 80 μM S18 (right). The figure shows phase contrast and overlay with RFP (red) fluorescence. Arrow in A indicates apoptotic cells.
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fig5: Alteration of neural stem cell apoptosis by antisense knockdown or overexpression of PAR-4. (A) Differentiating ES cells at the NP1 stage were transfected with a morpholino phosphorodiamidate antisense oligonucleotide against PAR-4, and 48 h later (NP3 stage), were incubated overnight with 80 μM S18. The figure shows PAR-4 (red), nestin (green), and TUNEL (blue) staining of cells transfected with standard control antisense oligonucleotide (left), and cells transfected with PAR-4–specific antisense oligonucleotide followed by incubation with S18 (right). (B) Staining of PAR-4 on immunoblots of protein from differentiating ES cells (NP3 stage) that were transfected with or without PAR-4–specific antisense oligonucleotide or PAR-4-RFP, respectively. Lane 1, untransfected (UT) NP cells; lane 2, NP cells transfected with standard control antisense oligonucleotide (Con); lane 3, NP cells transfected with PAR-4–specific antisense oligonucleotide (Anti); lane 4, NP cells transfected with PAR-4-RFP. (C) In differentiating ES cells at the EB8 stage, inhibition of ceramide biosynthesis was initiated by incubation with 50 nM myriocin, and was then maintained throughout the subsequent differentiation stages. After NP expansion (NP1 stage), cells were transfected with PAR-4-RFP, and 48 h later (NP3 stage), were incubated overnight with 80 μM S18 (right). The figure shows phase contrast and overlay with RFP (red) fluorescence. Arrow in A indicates apoptotic cells.
Mentions: To suppress PAR-4 expression, ES cells at the NP1 stage were transfected with a PAR-4–specific morpholino phosphorodiamidate antisense oligonucleotide (morpholino) that was designed on the basis of a previously published antisense oligonucleotide sequence (Guo et al., 1998, 2001). Morpholinos have been shown to avoid many of the pitfalls associated with conventional antisense oligonucleotides, and have been successfully used for transfection of embryos and cultured cells (Morcos, 2001). Fig. 5 A (left) shows NP cells 48 h after transfection with a standard morpholino provided as negative control. The number of TUNEL-stained cells in the negative control was equivalent to that found with untransfected cells (35%, see Fig. 3 B), indicating that the degree of apoptosis was not affected due to any unspecific effect of the morpholino. This result was consistent with the observation that the expression level of PAR-4 in untransfected and control morpholino-transfected NP cells was the same (Fig. 5 B, lane 1 and lane 2). Incubation of control morpholino-transfected NP cells with 80 μM S18 increased the number of apoptotic cells to that found with S18-treated, untransfected cells (>80%). However, transfection of NP cells with a PAR-4–specific antisense morpholino reduced S18-induced apoptosis to a level <30% (Fig. 5 A, right). Consistently, transfection with the PAR-4 antisense morpholino suppressed the expression level of PAR-4 to ∼25% of that detected in untransfected cells (Fig. 5 B, lane 3).

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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