Limits...
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.

Show MeSH
In vitro neuronal differentiation of ES cells, overview, and marker protein expression. ES, embryonic stem cell; EB, embryoid body; NP, neuronal progenitor cell; GRP, glial restricted precursor cells; NRP, neuronal restricted precursor cell (for terminology, see Mayer-Proschel et al., 1997; Wu et al., 2002). In B, immunostaining for marker proteins was performed with protein extracts from stem cells at the differentiation stages shown in A.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172704&req=5

fig1: In vitro neuronal differentiation of ES cells, overview, and marker protein expression. ES, embryonic stem cell; EB, embryoid body; NP, neuronal progenitor cell; GRP, glial restricted precursor cells; NRP, neuronal restricted precursor cell (for terminology, see Mayer-Proschel et al., 1997; Wu et al., 2002). In B, immunostaining for marker proteins was performed with protein extracts from stem cells at the differentiation stages shown in A.

Mentions: Mouse ES cells were differentiated after a serum deprivation protocol outlined in Fig. 1 A. This method yielded ES-derived cell cultures highly enriched in neural cells after 25 d in culture (Okabe et al., 1996; Hancock et al., 2000). Neuronal differentiation was verified by staining of marker proteins using immunoblotting (Fig. 1 B) and immunofluorescence microscopy (Fig. 2). ES cell differentiation was initiated by aggregating the ES cells (Fig. 2 A) to form EBs. The EBs were incubated in suspension culture in serum-containing medium for 4 d (Fig. 1 A, stages EB1–EB4). The differentiating EBs were then plated on tissue culture plates and allowed to attach in serum-containing medium for 1 d, and were then shifted to serum-free medium for three additional days of culture (Fig. 1 A and Fig. 2 A, EB5–EB8). The Oct4 protein, a marker of pluripotent stem cells (Pesce and Scholer, 2001), was detected in the EB8 stage, reflecting the presence of residual undifferentiated pluripotent stem cells within the EBs (Fig. 1 B, lane 1). No Oct4 expression was detectable after the dissociation and replating of the EBs in serum-free, FGF-2–containing medium (Fig. 1, A and B; lanes 2–4). The serum-free conditions did not support the proliferation of nonneural cell types, whereas FGF-2 supported the robust proliferation of NP cells (Fig. 1 A, stages NP1–NP4; Fig. 2 B shows NP2). The early neural precursor cell marker vimentin was only detected in EB8 and NP2 (Fig. 1 B, lane 1 and lane 2), whereas the NP marker nestin was detected at low levels at EB8 and at high levels at NP2 and D1 (Fig. 1 B, lanes 1–3; Fig. 2 B). The expansion of EBs was followed by a massive increase of the number of nestin-positive progenitor cells from ∼60% (Fig. 2 B, NP2) to >80% at NP4. Differentiation of NP cells to glial cells and neurons was initiated by withdrawal of FGF-2 from the culture medium (Fig. 1 A; stages D1–D4) and verified by staining for the glial fibrillary acidic protein (GFAP) and the neuronal marker proteins microtubuli-associated protein 2 (MAP-2) and synaptophysin (Figs. 1 B, lane 3 and lane 4; Fig. 2 C). No expression of NP markers was detected after D1 (Fig. 1 B, lane 4). Expression of GFAP was detected at D1 and D4, whereas MAP-2 and synaptophysin were only detected at D4, the most mature differentiation stage tested. It should be noted that <20% of Hoechst-stained cells showed neither GFAP nor MAP-2 staining, which verifies that the portion of nonneuronal cells within the fully differentiated culture was negligibly low (Fig. 2 C).


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)

In vitro neuronal differentiation of ES cells, overview, and marker protein expression. ES, embryonic stem cell; EB, embryoid body; NP, neuronal progenitor cell; GRP, glial restricted precursor cells; NRP, neuronal restricted precursor cell (for terminology, see Mayer-Proschel et al., 1997; Wu et al., 2002). In B, immunostaining for marker proteins was performed with protein extracts from stem cells at the differentiation stages shown in A.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172704&req=5

fig1: In vitro neuronal differentiation of ES cells, overview, and marker protein expression. ES, embryonic stem cell; EB, embryoid body; NP, neuronal progenitor cell; GRP, glial restricted precursor cells; NRP, neuronal restricted precursor cell (for terminology, see Mayer-Proschel et al., 1997; Wu et al., 2002). In B, immunostaining for marker proteins was performed with protein extracts from stem cells at the differentiation stages shown in A.
Mentions: Mouse ES cells were differentiated after a serum deprivation protocol outlined in Fig. 1 A. This method yielded ES-derived cell cultures highly enriched in neural cells after 25 d in culture (Okabe et al., 1996; Hancock et al., 2000). Neuronal differentiation was verified by staining of marker proteins using immunoblotting (Fig. 1 B) and immunofluorescence microscopy (Fig. 2). ES cell differentiation was initiated by aggregating the ES cells (Fig. 2 A) to form EBs. The EBs were incubated in suspension culture in serum-containing medium for 4 d (Fig. 1 A, stages EB1–EB4). The differentiating EBs were then plated on tissue culture plates and allowed to attach in serum-containing medium for 1 d, and were then shifted to serum-free medium for three additional days of culture (Fig. 1 A and Fig. 2 A, EB5–EB8). The Oct4 protein, a marker of pluripotent stem cells (Pesce and Scholer, 2001), was detected in the EB8 stage, reflecting the presence of residual undifferentiated pluripotent stem cells within the EBs (Fig. 1 B, lane 1). No Oct4 expression was detectable after the dissociation and replating of the EBs in serum-free, FGF-2–containing medium (Fig. 1, A and B; lanes 2–4). The serum-free conditions did not support the proliferation of nonneural cell types, whereas FGF-2 supported the robust proliferation of NP cells (Fig. 1 A, stages NP1–NP4; Fig. 2 B shows NP2). The early neural precursor cell marker vimentin was only detected in EB8 and NP2 (Fig. 1 B, lane 1 and lane 2), whereas the NP marker nestin was detected at low levels at EB8 and at high levels at NP2 and D1 (Fig. 1 B, lanes 1–3; Fig. 2 B). The expansion of EBs was followed by a massive increase of the number of nestin-positive progenitor cells from ∼60% (Fig. 2 B, NP2) to >80% at NP4. Differentiation of NP cells to glial cells and neurons was initiated by withdrawal of FGF-2 from the culture medium (Fig. 1 A; stages D1–D4) and verified by staining for the glial fibrillary acidic protein (GFAP) and the neuronal marker proteins microtubuli-associated protein 2 (MAP-2) and synaptophysin (Figs. 1 B, lane 3 and lane 4; Fig. 2 C). No expression of NP markers was detected after D1 (Fig. 1 B, lane 4). Expression of GFAP was detected at D1 and D4, whereas MAP-2 and synaptophysin were only detected at D4, the most mature differentiation stage tested. It should be noted that <20% of Hoechst-stained cells showed neither GFAP nor MAP-2 staining, which verifies that the portion of nonneuronal cells within the fully differentiated culture was negligibly low (Fig. 2 C).

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.

Show MeSH