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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 and caspase-8 by endogenous FasL is required for death of motoneurons in the absence of trophic support. (A) Freshly purified rat motoneurons were induced to die by culturing them in basal medium only for 24 h, or were kept alive by addition of an optimal concentration of BDNF (1 ng/ml). Increasing concentrations of Fas-Fc (0.01–1 μg/ml), which inhibits Fas activation by FasL, added to basal medium promoted their survival (expressed as a percentage of the number in BDNF) in a dose-dependent manner. Addition of 1 μg/ml of Fas-Fc to motoneurons cultured in the presence of BDNF (1 ng/ml) had no effect on their survival. (B) Immunolabeling of two cultured E12.5 mouse motoneurons using a specific antibody recognizing all forms of caspase-8. Double-labeling using DAPI (data not shown) allowed for localization of nuclei (arrows). All motoneurons presented clear granular labeling of the cytoplasm. (C) After seeding, rat motoneurons were incubated with increasing doses of the caspase-8 peptide inhibitor IETD-fmk (0.01–1 μM) without or with BDNF (1 ng/ml). The number of surviving cells was measured 24 h later and expressed relative to survival in BDNF. (D) Western blotting reveals the presence of caspase-8 in ventral spinal cord from mouse embryos, and in purified mouse motoneurons. The molecular mass of the major species (43 kD) is significantly lower than in nonneural cell lines (53–55 kD).
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Figure 4: Activation of Fas and caspase-8 by endogenous FasL is required for death of motoneurons in the absence of trophic support. (A) Freshly purified rat motoneurons were induced to die by culturing them in basal medium only for 24 h, or were kept alive by addition of an optimal concentration of BDNF (1 ng/ml). Increasing concentrations of Fas-Fc (0.01–1 μg/ml), which inhibits Fas activation by FasL, added to basal medium promoted their survival (expressed as a percentage of the number in BDNF) in a dose-dependent manner. Addition of 1 μg/ml of Fas-Fc to motoneurons cultured in the presence of BDNF (1 ng/ml) had no effect on their survival. (B) Immunolabeling of two cultured E12.5 mouse motoneurons using a specific antibody recognizing all forms of caspase-8. Double-labeling using DAPI (data not shown) allowed for localization of nuclei (arrows). All motoneurons presented clear granular labeling of the cytoplasm. (C) After seeding, rat motoneurons were incubated with increasing doses of the caspase-8 peptide inhibitor IETD-fmk (0.01–1 μM) without or with BDNF (1 ng/ml). The number of surviving cells was measured 24 h later and expressed relative to survival in BDNF. (D) Western blotting reveals the presence of caspase-8 in ventral spinal cord from mouse embryos, and in purified mouse motoneurons. The molecular mass of the major species (43 kD) is significantly lower than in nonneural cell lines (53–55 kD).

Mentions: Since motoneurons coexpressed the death receptor Fas together with its ligand, we asked whether Fas might play a role in motoneuron cell death. When purified rat motoneurons were grown in basal medium at low density (1,000 per 16-mm well) for 24 h, about half of them died (Fig. 4 A). Their death was completely prevented by BDNF (1 ng/ml). Fas-Fc, which contains the extracellular domain of Fas fused to an Ig Fc domain, is an antagonist known to block activation of Fas by FasL. Fas-Fc inhibited ∼75% of the death of trophically deprived motoneurons in a dose-dependent manner (Fig. 4 A). Higher doses of Fas-Fc were slightly less efficient (data not shown), but this may have been due to toxicity of the reagent. This suggested that endogenous FasL was activating Fas in these neurons, thereby triggering the cell death process.


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 and caspase-8 by endogenous FasL is required for death of motoneurons in the absence of trophic support. (A) Freshly purified rat motoneurons were induced to die by culturing them in basal medium only for 24 h, or were kept alive by addition of an optimal concentration of BDNF (1 ng/ml). Increasing concentrations of Fas-Fc (0.01–1 μg/ml), which inhibits Fas activation by FasL, added to basal medium promoted their survival (expressed as a percentage of the number in BDNF) in a dose-dependent manner. Addition of 1 μg/ml of Fas-Fc to motoneurons cultured in the presence of BDNF (1 ng/ml) had no effect on their survival. (B) Immunolabeling of two cultured E12.5 mouse motoneurons using a specific antibody recognizing all forms of caspase-8. Double-labeling using DAPI (data not shown) allowed for localization of nuclei (arrows). All motoneurons presented clear granular labeling of the cytoplasm. (C) After seeding, rat motoneurons were incubated with increasing doses of the caspase-8 peptide inhibitor IETD-fmk (0.01–1 μM) without or with BDNF (1 ng/ml). The number of surviving cells was measured 24 h later and expressed relative to survival in BDNF. (D) Western blotting reveals the presence of caspase-8 in ventral spinal cord from mouse embryos, and in purified mouse motoneurons. The molecular mass of the major species (43 kD) is significantly lower than in nonneural cell lines (53–55 kD).
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Related In: Results  -  Collection

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Figure 4: Activation of Fas and caspase-8 by endogenous FasL is required for death of motoneurons in the absence of trophic support. (A) Freshly purified rat motoneurons were induced to die by culturing them in basal medium only for 24 h, or were kept alive by addition of an optimal concentration of BDNF (1 ng/ml). Increasing concentrations of Fas-Fc (0.01–1 μg/ml), which inhibits Fas activation by FasL, added to basal medium promoted their survival (expressed as a percentage of the number in BDNF) in a dose-dependent manner. Addition of 1 μg/ml of Fas-Fc to motoneurons cultured in the presence of BDNF (1 ng/ml) had no effect on their survival. (B) Immunolabeling of two cultured E12.5 mouse motoneurons using a specific antibody recognizing all forms of caspase-8. Double-labeling using DAPI (data not shown) allowed for localization of nuclei (arrows). All motoneurons presented clear granular labeling of the cytoplasm. (C) After seeding, rat motoneurons were incubated with increasing doses of the caspase-8 peptide inhibitor IETD-fmk (0.01–1 μM) without or with BDNF (1 ng/ml). The number of surviving cells was measured 24 h later and expressed relative to survival in BDNF. (D) Western blotting reveals the presence of caspase-8 in ventral spinal cord from mouse embryos, and in purified mouse motoneurons. The molecular mass of the major species (43 kD) is significantly lower than in nonneural cell lines (53–55 kD).
Mentions: Since motoneurons coexpressed the death receptor Fas together with its ligand, we asked whether Fas might play a role in motoneuron cell death. When purified rat motoneurons were grown in basal medium at low density (1,000 per 16-mm well) for 24 h, about half of them died (Fig. 4 A). Their death was completely prevented by BDNF (1 ng/ml). Fas-Fc, which contains the extracellular domain of Fas fused to an Ig Fc domain, is an antagonist known to block activation of Fas by FasL. Fas-Fc inhibited ∼75% of the death of trophically deprived motoneurons in a dose-dependent manner (Fig. 4 A). Higher doses of Fas-Fc were slightly less efficient (data not shown), but this may have been due to toxicity of the reagent. This suggested that endogenous FasL was activating Fas in these neurons, thereby triggering the cell death process.

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