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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.

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Activation of Fas triggers PCD of motoneurons even in the presence of optimal trophic support. Different agents known to activate Fas receptor by clustering were tested for their ability to trigger motoneuron cell death. (A) Purified E12.5 mouse motoneurons were cultured in the presence of BDNF (1 ng/ml), CT-1 (10 ng/ml), and GDNF (0.1 ng/ml) (+NTFs) for 1 d. Subsequently, the indicated concentrations of anti-Fas antibody were added in the continued presence of NTFs, and motoneuron survival was counted 2 d later, at 3 DIV. Counts were expressed relative to the value for NTFs at 3 DIV. Fas antibody induced a dose-dependent loss of 45% of the motoneurons. (B) An analogous experiment using purified motoneurons from E14 rat, cultured in the continued presence of BDNF (1 ng/ml). After 1 DIV, tagged soluble FasL was added at the indicated concentrations in the presence of 1 μg/ml of enhancer antibody, which had no effect when tested alone. Survival was counted 2 d later. (C) Comparison of the fractions of E14 rat motoneurons lost after 2 d of incubation with 10 ng/ml sFasL, in the presence of indicated neurotrophic factors used at the same concentrations as for mouse motoneurons in A. The number of motoneurons lost was expressed as a percentage of the total number of motoneurons present in the same conditions but without sFasL. All histograms are representative of at least three independent experiments. Error bars represent the mean ± range of duplicate wells.
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Figure 5: Activation of Fas triggers PCD of motoneurons even in the presence of optimal trophic support. Different agents known to activate Fas receptor by clustering were tested for their ability to trigger motoneuron cell death. (A) Purified E12.5 mouse motoneurons were cultured in the presence of BDNF (1 ng/ml), CT-1 (10 ng/ml), and GDNF (0.1 ng/ml) (+NTFs) for 1 d. Subsequently, the indicated concentrations of anti-Fas antibody were added in the continued presence of NTFs, and motoneuron survival was counted 2 d later, at 3 DIV. Counts were expressed relative to the value for NTFs at 3 DIV. Fas antibody induced a dose-dependent loss of 45% of the motoneurons. (B) An analogous experiment using purified motoneurons from E14 rat, cultured in the continued presence of BDNF (1 ng/ml). After 1 DIV, tagged soluble FasL was added at the indicated concentrations in the presence of 1 μg/ml of enhancer antibody, which had no effect when tested alone. Survival was counted 2 d later. (C) Comparison of the fractions of E14 rat motoneurons lost after 2 d of incubation with 10 ng/ml sFasL, in the presence of indicated neurotrophic factors used at the same concentrations as for mouse motoneurons in A. The number of motoneurons lost was expressed as a percentage of the total number of motoneurons present in the same conditions but without sFasL. All histograms are representative of at least three independent experiments. Error bars represent the mean ± range of duplicate wells.

Mentions: Motoneurons purified from E12.5 mouse embryos were cultured in the presence of a cocktail of trophic factors (BDNF, CNTF, and GDNF) at concentrations determined previously to ensure optimal survival (Arce et al. 1999). After 24 h to allow attachment and initial neurite outgrowth, antibody to the extracellular domain of mouse Fas was added in the continued presence of trophic factors to induce clustering of Fas. 2 d later (i.e., after 3 d in vitro [DIV]), anti-Fas antibody had caused dose-dependent motoneuron loss, up to a maximum reduction of 45% using 10 ng/ml antibody (Fig. 5 A). The half-maximal dose of antibody required to induce death of motoneurons (0.7 ng/ml) is close to that reported for lymphoid cells (Watanabe-Fukunaga et al. 1992). Control antibodies including the enhancer antibody (see below) had no effect on survival (data not shown).


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

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

Activation of Fas triggers PCD of motoneurons even in the presence of optimal trophic support. Different agents known to activate Fas receptor by clustering were tested for their ability to trigger motoneuron cell death. (A) Purified E12.5 mouse motoneurons were cultured in the presence of BDNF (1 ng/ml), CT-1 (10 ng/ml), and GDNF (0.1 ng/ml) (+NTFs) for 1 d. Subsequently, the indicated concentrations of anti-Fas antibody were added in the continued presence of NTFs, and motoneuron survival was counted 2 d later, at 3 DIV. Counts were expressed relative to the value for NTFs at 3 DIV. Fas antibody induced a dose-dependent loss of 45% of the motoneurons. (B) An analogous experiment using purified motoneurons from E14 rat, cultured in the continued presence of BDNF (1 ng/ml). After 1 DIV, tagged soluble FasL was added at the indicated concentrations in the presence of 1 μg/ml of enhancer antibody, which had no effect when tested alone. Survival was counted 2 d later. (C) Comparison of the fractions of E14 rat motoneurons lost after 2 d of incubation with 10 ng/ml sFasL, in the presence of indicated neurotrophic factors used at the same concentrations as for mouse motoneurons in A. The number of motoneurons lost was expressed as a percentage of the total number of motoneurons present in the same conditions but without sFasL. All histograms are representative of at least three independent experiments. Error bars represent the mean ± range of duplicate wells.
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Figure 5: Activation of Fas triggers PCD of motoneurons even in the presence of optimal trophic support. Different agents known to activate Fas receptor by clustering were tested for their ability to trigger motoneuron cell death. (A) Purified E12.5 mouse motoneurons were cultured in the presence of BDNF (1 ng/ml), CT-1 (10 ng/ml), and GDNF (0.1 ng/ml) (+NTFs) for 1 d. Subsequently, the indicated concentrations of anti-Fas antibody were added in the continued presence of NTFs, and motoneuron survival was counted 2 d later, at 3 DIV. Counts were expressed relative to the value for NTFs at 3 DIV. Fas antibody induced a dose-dependent loss of 45% of the motoneurons. (B) An analogous experiment using purified motoneurons from E14 rat, cultured in the continued presence of BDNF (1 ng/ml). After 1 DIV, tagged soluble FasL was added at the indicated concentrations in the presence of 1 μg/ml of enhancer antibody, which had no effect when tested alone. Survival was counted 2 d later. (C) Comparison of the fractions of E14 rat motoneurons lost after 2 d of incubation with 10 ng/ml sFasL, in the presence of indicated neurotrophic factors used at the same concentrations as for mouse motoneurons in A. The number of motoneurons lost was expressed as a percentage of the total number of motoneurons present in the same conditions but without sFasL. All histograms are representative of at least three independent experiments. Error bars represent the mean ± range of duplicate wells.
Mentions: Motoneurons purified from E12.5 mouse embryos were cultured in the presence of a cocktail of trophic factors (BDNF, CNTF, and GDNF) at concentrations determined previously to ensure optimal survival (Arce et al. 1999). After 24 h to allow attachment and initial neurite outgrowth, antibody to the extracellular domain of mouse Fas was added in the continued presence of trophic factors to induce clustering of Fas. 2 d later (i.e., after 3 d in vitro [DIV]), anti-Fas antibody had caused dose-dependent motoneuron loss, up to a maximum reduction of 45% using 10 ng/ml antibody (Fig. 5 A). The half-maximal dose of antibody required to induce death of motoneurons (0.7 ng/ml) is close to that reported for lymphoid cells (Watanabe-Fukunaga et al. 1992). Control antibodies including the enhancer antibody (see below) had no effect on survival (data not shown).

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