Limits...
Programmed cell death of embryonic motoneurons triggered through the Fas death receptor.

Raoul C, Henderson CE, Pettmann B - J. Cell Biol. (1999)

Bottom Line: Sensitivity to Fas activation is regulated: motoneurons cultured for 3 d with neurotrophic factors became completely resistant.Motoneurons resistant to Fas activation expressed high levels of FLICE-inhibitory protein (FLIP), an endogenous inhibitor of caspase-8 activation.Our results suggest that Fas can act as a driving force for motoneuron PCD, and raise the possibility that active triggering of PCD may contribute to motoneuron loss during normal development and/or in pathological situations.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale U.382, Developmental Biology Institute of Marseille (CNRS), Institut National de la Santé et de la Recherche Médicale, Université de la Mediterranee, AP Marseille, France.

ABSTRACT
About 50% of spinal motoneurons undergo programmed cell death (PCD) after target contact, but little is known about how this process is initiated. Embryonic motoneurons coexpress the death receptor Fas and its ligand FasL at the stage at which PCD is about to begin. In the absence of trophic factors, many motoneurons die in culture within 2 d. Most (75%) of these were saved by Fas-Fc receptor body, which blocks interactions between Fas and FasL, or by the caspase-8 inhibitor tetrapeptide IETD. Therefore, activation of Fas by endogenous FasL underlies cell death induced by trophic deprivation. In the presence of neurotrophic factors, exogenous Fas activators such as soluble FasL or anti-Fas antibodies triggered PCD of 40-50% of purified motoneurons over the following 3-5 d; this treatment led to activation of caspase-3, and was blocked by IETD. Sensitivity to Fas activation is regulated: motoneurons cultured for 3 d with neurotrophic factors became completely resistant. Levels of Fas expressed by motoneurons varied little, but FasL was upregulated in the absence of neurotrophic factors. Motoneurons resistant to Fas activation expressed high levels of FLICE-inhibitory protein (FLIP), an endogenous inhibitor of caspase-8 activation. Our results suggest that Fas can act as a driving force for motoneuron PCD, and raise the possibility that active triggering of PCD may contribute to motoneuron loss during normal development and/or in pathological situations.

Show MeSH

Related in: MedlinePlus

Resistance of motoneurons to the cell-killing effects of Fas activation is tightly regulated. Mouse motoneurons were cultured in the continued presence of BDNF, CNTF, and GDNF (NTFs) and then treated with the Fas activators sFasL (50 ng/ml) or anti-Fas antibodies (50 ng/ml). Motoneuron survival in all experiments was counted at 5 DIV, and expressed relative to the value in NTFs alone at that time. (A) Fas activators were added at 1 DIV only. (B) Fas activators were added at 1 and 3 DIV; no further motoneuron death was observed. (C) Fas activators were added at 3 DIV: all motoneurons became resistant to Fas activation. Histograms are representative of similar results obtained in three independent experiments. Error bars represent the mean ± range of duplicate values.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169347&req=5

Figure 7: Resistance of motoneurons to the cell-killing effects of Fas activation is tightly regulated. Mouse motoneurons were cultured in the continued presence of BDNF, CNTF, and GDNF (NTFs) and then treated with the Fas activators sFasL (50 ng/ml) or anti-Fas antibodies (50 ng/ml). Motoneuron survival in all experiments was counted at 5 DIV, and expressed relative to the value in NTFs alone at that time. (A) Fas activators were added at 1 DIV only. (B) Fas activators were added at 1 and 3 DIV; no further motoneuron death was observed. (C) Fas activators were added at 3 DIV: all motoneurons became resistant to Fas activation. Histograms are representative of similar results obtained in three independent experiments. Error bars represent the mean ± range of duplicate values.

Mentions: It was striking (Fig. 5) that ∼50% of motoneurons were resistant to the effects of exogenous Fas activation in the presence of trophic factors. We tested the survival of mouse motoneurons treated at different stages with Fas activators (Fig. 7). In a first experiment analogous to those in Fig. 5, mouse motoneurons were seeded in a cocktail of neurotrophic factors, treated with either sFasL or anti-Fas at 1 DIV, and their survival was counted 4 d later, at 5 DIV (Fig. 7 A). Some motoneurons (∼10%) died over the 5-d culture period even in the presence of trophic factors, and so values were expressed as a percentage of the value at 5 DIV with trophic factors alone. In these conditions, Fas activation led to loss of 50% of motoneurons. To rule out the possibility that incomplete motoneuron loss reflected instability of reagents in the culture dish, we then added Fas activators at both 1 and 3 DIV, and counted survival at 5 DIV (Fig. 7 B). No further motoneuron loss was observed, demonstrating that some motoneurons are truly resistant to Fas activation. We then asked whether this resistance might be regulated in vitro, by first culturing motoneurons for 3 DIV in the presence of trophic factors, then adding Fas activators, and counting survival at 5 DIV. Surprisingly, no motoneuron death at all was triggered in these conditions (Fig. 7 C).


Programmed cell death of embryonic motoneurons triggered through the Fas death receptor.

Raoul C, Henderson CE, Pettmann B - J. Cell Biol. (1999)

Resistance of motoneurons to the cell-killing effects of Fas activation is tightly regulated. Mouse motoneurons were cultured in the continued presence of BDNF, CNTF, and GDNF (NTFs) and then treated with the Fas activators sFasL (50 ng/ml) or anti-Fas antibodies (50 ng/ml). Motoneuron survival in all experiments was counted at 5 DIV, and expressed relative to the value in NTFs alone at that time. (A) Fas activators were added at 1 DIV only. (B) Fas activators were added at 1 and 3 DIV; no further motoneuron death was observed. (C) Fas activators were added at 3 DIV: all motoneurons became resistant to Fas activation. Histograms are representative of similar results obtained in three independent experiments. Error bars represent the mean ± range of duplicate values.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169347&req=5

Figure 7: Resistance of motoneurons to the cell-killing effects of Fas activation is tightly regulated. Mouse motoneurons were cultured in the continued presence of BDNF, CNTF, and GDNF (NTFs) and then treated with the Fas activators sFasL (50 ng/ml) or anti-Fas antibodies (50 ng/ml). Motoneuron survival in all experiments was counted at 5 DIV, and expressed relative to the value in NTFs alone at that time. (A) Fas activators were added at 1 DIV only. (B) Fas activators were added at 1 and 3 DIV; no further motoneuron death was observed. (C) Fas activators were added at 3 DIV: all motoneurons became resistant to Fas activation. Histograms are representative of similar results obtained in three independent experiments. Error bars represent the mean ± range of duplicate values.
Mentions: It was striking (Fig. 5) that ∼50% of motoneurons were resistant to the effects of exogenous Fas activation in the presence of trophic factors. We tested the survival of mouse motoneurons treated at different stages with Fas activators (Fig. 7). In a first experiment analogous to those in Fig. 5, mouse motoneurons were seeded in a cocktail of neurotrophic factors, treated with either sFasL or anti-Fas at 1 DIV, and their survival was counted 4 d later, at 5 DIV (Fig. 7 A). Some motoneurons (∼10%) died over the 5-d culture period even in the presence of trophic factors, and so values were expressed as a percentage of the value at 5 DIV with trophic factors alone. In these conditions, Fas activation led to loss of 50% of motoneurons. To rule out the possibility that incomplete motoneuron loss reflected instability of reagents in the culture dish, we then added Fas activators at both 1 and 3 DIV, and counted survival at 5 DIV (Fig. 7 B). No further motoneuron loss was observed, demonstrating that some motoneurons are truly resistant to Fas activation. We then asked whether this resistance might be regulated in vitro, by first culturing motoneurons for 3 DIV in the presence of trophic factors, then adding Fas activators, and counting survival at 5 DIV. Surprisingly, no motoneuron death at all was triggered in these conditions (Fig. 7 C).

Bottom Line: Sensitivity to Fas activation is regulated: motoneurons cultured for 3 d with neurotrophic factors became completely resistant.Motoneurons resistant to Fas activation expressed high levels of FLICE-inhibitory protein (FLIP), an endogenous inhibitor of caspase-8 activation.Our results suggest that Fas can act as a driving force for motoneuron PCD, and raise the possibility that active triggering of PCD may contribute to motoneuron loss during normal development and/or in pathological situations.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale U.382, Developmental Biology Institute of Marseille (CNRS), Institut National de la Santé et de la Recherche Médicale, Université de la Mediterranee, AP Marseille, France.

ABSTRACT
About 50% of spinal motoneurons undergo programmed cell death (PCD) after target contact, but little is known about how this process is initiated. Embryonic motoneurons coexpress the death receptor Fas and its ligand FasL at the stage at which PCD is about to begin. In the absence of trophic factors, many motoneurons die in culture within 2 d. Most (75%) of these were saved by Fas-Fc receptor body, which blocks interactions between Fas and FasL, or by the caspase-8 inhibitor tetrapeptide IETD. Therefore, activation of Fas by endogenous FasL underlies cell death induced by trophic deprivation. In the presence of neurotrophic factors, exogenous Fas activators such as soluble FasL or anti-Fas antibodies triggered PCD of 40-50% of purified motoneurons over the following 3-5 d; this treatment led to activation of caspase-3, and was blocked by IETD. Sensitivity to Fas activation is regulated: motoneurons cultured for 3 d with neurotrophic factors became completely resistant. Levels of Fas expressed by motoneurons varied little, but FasL was upregulated in the absence of neurotrophic factors. Motoneurons resistant to Fas activation expressed high levels of FLICE-inhibitory protein (FLIP), an endogenous inhibitor of caspase-8 activation. Our results suggest that Fas can act as a driving force for motoneuron PCD, and raise the possibility that active triggering of PCD may contribute to motoneuron loss during normal development and/or in pathological situations.

Show MeSH
Related in: MedlinePlus