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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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Motoneuron cell death triggered by Fas activation in the presence of trophic factors involves caspases-3 and -8. (A) Activation of caspase-3 in cultured mouse motoneurons treated with anti-Fas was visualized using the CM1 antibody, which specifically recognizes the activated form of caspase-3. The motoneuron illustrated was undergoing apoptosis, as visualized by the fragmented chromatin of the nucleus stained with DAPI (inset). Note the intense perinuclear staining for activated caspase-3. (B) Increase in the fraction of CM1-positive mouse motoneurons 30 h after treatment with anti-Fas antibodies in the presence of neurotrophic factors. After immunostaining, CM1-positive motoneurons were counted along two diameters of 14-mm coverslips (∼100 motoneurons counted for each). All CM1-positive cells showed pyknotic nuclei by DAPI staining. Values are means ± SEM of five coverslips, and are typical of two independent experiments (asterisks indicate P = 0.001 by t test). Total survival was not significantly different in the two conditions (P > 0.5; data not shown). (C) Death of E14 rat motoneurons triggered by sFasL (10 ng/ml) in the presence of BDNF (1 ng/ml) is inhibited in a dose-dependent fashion by the caspase-3 peptide inhibitor DEVD-fmk. (D) Death of mouse motoneurons triggered by sFasL (10 ng/ml) and enhancer (1 μg/ml), or anti-Fas antibodies (10 ng/ml), is blocked by DEVD-fmk (10 μM). Survival is expressed as the percentage of the number of motoneurons surviving in the presence of BDNF alone. (E) Death of rat motoneurons triggered by sFasL (10 ng/ml, in the presence of enhancer) is inhibited by the caspase-8 inhibitor IETD-fmk (1 μM). C, D, and E show combined mean values (± S.D.) from duplicate wells in two independent experiments.
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Figure 6: Motoneuron cell death triggered by Fas activation in the presence of trophic factors involves caspases-3 and -8. (A) Activation of caspase-3 in cultured mouse motoneurons treated with anti-Fas was visualized using the CM1 antibody, which specifically recognizes the activated form of caspase-3. The motoneuron illustrated was undergoing apoptosis, as visualized by the fragmented chromatin of the nucleus stained with DAPI (inset). Note the intense perinuclear staining for activated caspase-3. (B) Increase in the fraction of CM1-positive mouse motoneurons 30 h after treatment with anti-Fas antibodies in the presence of neurotrophic factors. After immunostaining, CM1-positive motoneurons were counted along two diameters of 14-mm coverslips (∼100 motoneurons counted for each). All CM1-positive cells showed pyknotic nuclei by DAPI staining. Values are means ± SEM of five coverslips, and are typical of two independent experiments (asterisks indicate P = 0.001 by t test). Total survival was not significantly different in the two conditions (P > 0.5; data not shown). (C) Death of E14 rat motoneurons triggered by sFasL (10 ng/ml) in the presence of BDNF (1 ng/ml) is inhibited in a dose-dependent fashion by the caspase-3 peptide inhibitor DEVD-fmk. (D) Death of mouse motoneurons triggered by sFasL (10 ng/ml) and enhancer (1 μg/ml), or anti-Fas antibodies (10 ng/ml), is blocked by DEVD-fmk (10 μM). Survival is expressed as the percentage of the number of motoneurons surviving in the presence of BDNF alone. (E) Death of rat motoneurons triggered by sFasL (10 ng/ml, in the presence of enhancer) is inhibited by the caspase-8 inhibitor IETD-fmk (1 μM). C, D, and E show combined mean values (± S.D.) from duplicate wells in two independent experiments.

Mentions: We first focused on caspase-3, a downstream caspase known to be required for PCD of many neurons (Kuida et al. 1996), and of motoneurons in particular (Li et al. 1998). Using an antibody (CM1) that specifically recognizes the activated form of caspase-3 (Srinivasan et al. 1998b), we showed that many cultured motoneurons exposed to Fas activators expressed activated caspase-3, and that this reactivity was especially strong in the motoneurons with apoptotic nuclei that had not yet detached from the culture dish (Fig. 6 A). We quantified CM1-positive motoneurons 30 h after treatment (or not) with anti-Fas antibodies (Fig. 6 B). At this stage, there was not yet a significant difference in total motoneuron numbers in the presence or absence of anti-Fas (P > 0.5; data not shown). Without Fas activator, 8.0 ± 0.9% (mean ± SEM; n = 5) of motoneurons expressed intense CM1 immunoreactivity and showed fragmented chromatin using Hoechst staining. Using anti-Fas antibodies, this value was significantly increased (19.0 ± 1.6%; P = 0.001 by t test). To test the functional significance of caspase-3 activation, we used the active-site peptide DEVD, a potent cell-permeable caspase inhibitor that is selective for caspase-3 (Nicholson et al. 1995; Villa et al. 1997), but can also inhibit caspase-8 (Garcia-Calvo et al. 1998). Increasing concentrations of DEVD-fmk were added to rat motoneurons cultured in BDNF, 2 h before addition of sFasL (10 ng/ml) and enhancer antibody. Motoneuron loss was inhibited in a dose-dependent fashion (Fig. 6 C), and the concentration of DEVD required to completely prevent Fas-dependent motoneuron death (10 μM) was similar to or lower than those reported to prevent PCD in other cell types (Armstrong et al. 1997). DEVD was also able to completely inhibit the cell death of mouse motoneurons induced by sFasL or anti-Fas antibodies (Fig. 6 D). Thus, Fas most likely triggers PCD of motoneurons by a mechanism involving caspase-3.


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

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

Motoneuron cell death triggered by Fas activation in the presence of trophic factors involves caspases-3 and -8. (A) Activation of caspase-3 in cultured mouse motoneurons treated with anti-Fas was visualized using the CM1 antibody, which specifically recognizes the activated form of caspase-3. The motoneuron illustrated was undergoing apoptosis, as visualized by the fragmented chromatin of the nucleus stained with DAPI (inset). Note the intense perinuclear staining for activated caspase-3. (B) Increase in the fraction of CM1-positive mouse motoneurons 30 h after treatment with anti-Fas antibodies in the presence of neurotrophic factors. After immunostaining, CM1-positive motoneurons were counted along two diameters of 14-mm coverslips (∼100 motoneurons counted for each). All CM1-positive cells showed pyknotic nuclei by DAPI staining. Values are means ± SEM of five coverslips, and are typical of two independent experiments (asterisks indicate P = 0.001 by t test). Total survival was not significantly different in the two conditions (P > 0.5; data not shown). (C) Death of E14 rat motoneurons triggered by sFasL (10 ng/ml) in the presence of BDNF (1 ng/ml) is inhibited in a dose-dependent fashion by the caspase-3 peptide inhibitor DEVD-fmk. (D) Death of mouse motoneurons triggered by sFasL (10 ng/ml) and enhancer (1 μg/ml), or anti-Fas antibodies (10 ng/ml), is blocked by DEVD-fmk (10 μM). Survival is expressed as the percentage of the number of motoneurons surviving in the presence of BDNF alone. (E) Death of rat motoneurons triggered by sFasL (10 ng/ml, in the presence of enhancer) is inhibited by the caspase-8 inhibitor IETD-fmk (1 μM). C, D, and E show combined mean values (± S.D.) from duplicate wells in two independent experiments.
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Related In: Results  -  Collection

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Figure 6: Motoneuron cell death triggered by Fas activation in the presence of trophic factors involves caspases-3 and -8. (A) Activation of caspase-3 in cultured mouse motoneurons treated with anti-Fas was visualized using the CM1 antibody, which specifically recognizes the activated form of caspase-3. The motoneuron illustrated was undergoing apoptosis, as visualized by the fragmented chromatin of the nucleus stained with DAPI (inset). Note the intense perinuclear staining for activated caspase-3. (B) Increase in the fraction of CM1-positive mouse motoneurons 30 h after treatment with anti-Fas antibodies in the presence of neurotrophic factors. After immunostaining, CM1-positive motoneurons were counted along two diameters of 14-mm coverslips (∼100 motoneurons counted for each). All CM1-positive cells showed pyknotic nuclei by DAPI staining. Values are means ± SEM of five coverslips, and are typical of two independent experiments (asterisks indicate P = 0.001 by t test). Total survival was not significantly different in the two conditions (P > 0.5; data not shown). (C) Death of E14 rat motoneurons triggered by sFasL (10 ng/ml) in the presence of BDNF (1 ng/ml) is inhibited in a dose-dependent fashion by the caspase-3 peptide inhibitor DEVD-fmk. (D) Death of mouse motoneurons triggered by sFasL (10 ng/ml) and enhancer (1 μg/ml), or anti-Fas antibodies (10 ng/ml), is blocked by DEVD-fmk (10 μM). Survival is expressed as the percentage of the number of motoneurons surviving in the presence of BDNF alone. (E) Death of rat motoneurons triggered by sFasL (10 ng/ml, in the presence of enhancer) is inhibited by the caspase-8 inhibitor IETD-fmk (1 μM). C, D, and E show combined mean values (± S.D.) from duplicate wells in two independent experiments.
Mentions: We first focused on caspase-3, a downstream caspase known to be required for PCD of many neurons (Kuida et al. 1996), and of motoneurons in particular (Li et al. 1998). Using an antibody (CM1) that specifically recognizes the activated form of caspase-3 (Srinivasan et al. 1998b), we showed that many cultured motoneurons exposed to Fas activators expressed activated caspase-3, and that this reactivity was especially strong in the motoneurons with apoptotic nuclei that had not yet detached from the culture dish (Fig. 6 A). We quantified CM1-positive motoneurons 30 h after treatment (or not) with anti-Fas antibodies (Fig. 6 B). At this stage, there was not yet a significant difference in total motoneuron numbers in the presence or absence of anti-Fas (P > 0.5; data not shown). Without Fas activator, 8.0 ± 0.9% (mean ± SEM; n = 5) of motoneurons expressed intense CM1 immunoreactivity and showed fragmented chromatin using Hoechst staining. Using anti-Fas antibodies, this value was significantly increased (19.0 ± 1.6%; P = 0.001 by t test). To test the functional significance of caspase-3 activation, we used the active-site peptide DEVD, a potent cell-permeable caspase inhibitor that is selective for caspase-3 (Nicholson et al. 1995; Villa et al. 1997), but can also inhibit caspase-8 (Garcia-Calvo et al. 1998). Increasing concentrations of DEVD-fmk were added to rat motoneurons cultured in BDNF, 2 h before addition of sFasL (10 ng/ml) and enhancer antibody. Motoneuron loss was inhibited in a dose-dependent fashion (Fig. 6 C), and the concentration of DEVD required to completely prevent Fas-dependent motoneuron death (10 μM) was similar to or lower than those reported to prevent PCD in other cell types (Armstrong et al. 1997). DEVD was also able to completely inhibit the cell death of mouse motoneurons induced by sFasL or anti-Fas antibodies (Fig. 6 D). Thus, Fas most likely triggers PCD of motoneurons by a mechanism involving caspase-3.

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