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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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Regulation of FasL and FLIP by neurotrophic factors. (A and B) Levels of molecules involved in Fas signaling were followed using semiquantitative RT-PCR on motoneurons cultured for 1 and 3 DIV in the presence or absence of neurotrophic factors. (A) FasL mRNA is upregulated in motoneurons cultured in the absence of trophic factors. (B) Regulation of fas and FLIP mRNAs. mRNA samples were incubated with (+) or without (−) reverse transcriptase. Fas was expressed at relatively constant levels when normalized to actin, whereas FLIP was strongly upregulated in motoneurons cultured for 3 DIV in the presence of NTFs. (C) Strong upregulation of FLIP protein in the presence of neurotrophic factors. Western blots are shown of extracts of purified motoneurons cultured for 1 and 3 DIV in the presence of NTFs; two major forms of FLIP at 55 and 52 kD are observed. The loading control was neurofilament-68. (D) Immunolabeling of Fas-resistant mouse motoneurons cultured for 3 d in the presence of NTFs, using the blot-purified polyclonal antibodies against Fas characterized in Fig. 2. Fas continues to be expressed at the cell membrane. (E) Motoneurons treated for 3 d with NTFs also continue to express caspase-8. (F) Summary diagram showing the potential involvement of the Fas system in PCD of motoneurons. The main experimental strategies adopted in our study of the role of Fas are depicted. In addition, we have indicated the three levels at which neurotrophic factors may potentially act to block Fas-related motoneuron death.
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Figure 8: Regulation of FasL and FLIP by neurotrophic factors. (A and B) Levels of molecules involved in Fas signaling were followed using semiquantitative RT-PCR on motoneurons cultured for 1 and 3 DIV in the presence or absence of neurotrophic factors. (A) FasL mRNA is upregulated in motoneurons cultured in the absence of trophic factors. (B) Regulation of fas and FLIP mRNAs. mRNA samples were incubated with (+) or without (−) reverse transcriptase. Fas was expressed at relatively constant levels when normalized to actin, whereas FLIP was strongly upregulated in motoneurons cultured for 3 DIV in the presence of NTFs. (C) Strong upregulation of FLIP protein in the presence of neurotrophic factors. Western blots are shown of extracts of purified motoneurons cultured for 1 and 3 DIV in the presence of NTFs; two major forms of FLIP at 55 and 52 kD are observed. The loading control was neurofilament-68. (D) Immunolabeling of Fas-resistant mouse motoneurons cultured for 3 d in the presence of NTFs, using the blot-purified polyclonal antibodies against Fas characterized in Fig. 2. Fas continues to be expressed at the cell membrane. (E) Motoneurons treated for 3 d with NTFs also continue to express caspase-8. (F) Summary diagram showing the potential involvement of the Fas system in PCD of motoneurons. The main experimental strategies adopted in our study of the role of Fas are depicted. In addition, we have indicated the three levels at which neurotrophic factors may potentially act to block Fas-related motoneuron death.

Mentions: First we asked whether trophic deprivation led to upregulation of components of the Fas system. Levels of Fas, normalized to actin, were relatively unaffected by the presence or absence of trophic factors (Fig. 8 B). In contrast, whereas levels of FasL steadily decreased in motoneurons cultured with trophic factors, they were strongly upregulated in trophically deprived motoneurons (Fig. 8 A). Thus, regulation of levels of FasL may underlie the dependence of motoneurons on external trophic support.


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

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

Regulation of FasL and FLIP by neurotrophic factors. (A and B) Levels of molecules involved in Fas signaling were followed using semiquantitative RT-PCR on motoneurons cultured for 1 and 3 DIV in the presence or absence of neurotrophic factors. (A) FasL mRNA is upregulated in motoneurons cultured in the absence of trophic factors. (B) Regulation of fas and FLIP mRNAs. mRNA samples were incubated with (+) or without (−) reverse transcriptase. Fas was expressed at relatively constant levels when normalized to actin, whereas FLIP was strongly upregulated in motoneurons cultured for 3 DIV in the presence of NTFs. (C) Strong upregulation of FLIP protein in the presence of neurotrophic factors. Western blots are shown of extracts of purified motoneurons cultured for 1 and 3 DIV in the presence of NTFs; two major forms of FLIP at 55 and 52 kD are observed. The loading control was neurofilament-68. (D) Immunolabeling of Fas-resistant mouse motoneurons cultured for 3 d in the presence of NTFs, using the blot-purified polyclonal antibodies against Fas characterized in Fig. 2. Fas continues to be expressed at the cell membrane. (E) Motoneurons treated for 3 d with NTFs also continue to express caspase-8. (F) Summary diagram showing the potential involvement of the Fas system in PCD of motoneurons. The main experimental strategies adopted in our study of the role of Fas are depicted. In addition, we have indicated the three levels at which neurotrophic factors may potentially act to block Fas-related motoneuron death.
© Copyright Policy
Related In: Results  -  Collection

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Figure 8: Regulation of FasL and FLIP by neurotrophic factors. (A and B) Levels of molecules involved in Fas signaling were followed using semiquantitative RT-PCR on motoneurons cultured for 1 and 3 DIV in the presence or absence of neurotrophic factors. (A) FasL mRNA is upregulated in motoneurons cultured in the absence of trophic factors. (B) Regulation of fas and FLIP mRNAs. mRNA samples were incubated with (+) or without (−) reverse transcriptase. Fas was expressed at relatively constant levels when normalized to actin, whereas FLIP was strongly upregulated in motoneurons cultured for 3 DIV in the presence of NTFs. (C) Strong upregulation of FLIP protein in the presence of neurotrophic factors. Western blots are shown of extracts of purified motoneurons cultured for 1 and 3 DIV in the presence of NTFs; two major forms of FLIP at 55 and 52 kD are observed. The loading control was neurofilament-68. (D) Immunolabeling of Fas-resistant mouse motoneurons cultured for 3 d in the presence of NTFs, using the blot-purified polyclonal antibodies against Fas characterized in Fig. 2. Fas continues to be expressed at the cell membrane. (E) Motoneurons treated for 3 d with NTFs also continue to express caspase-8. (F) Summary diagram showing the potential involvement of the Fas system in PCD of motoneurons. The main experimental strategies adopted in our study of the role of Fas are depicted. In addition, we have indicated the three levels at which neurotrophic factors may potentially act to block Fas-related motoneuron death.
Mentions: First we asked whether trophic deprivation led to upregulation of components of the Fas system. Levels of Fas, normalized to actin, were relatively unaffected by the presence or absence of trophic factors (Fig. 8 B). In contrast, whereas levels of FasL steadily decreased in motoneurons cultured with trophic factors, they were strongly upregulated in trophically deprived motoneurons (Fig. 8 A). Thus, regulation of levels of FasL may underlie the dependence of motoneurons on external trophic support.

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