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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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Embryonic motoneurons express Fas and FasL both in situ and after purification. Total RNA was prepared from the indicated rat tissues, and RT-PCR analysis followed by Southern blotting was performed using specific primers and internal probes for FasL (A) and Fas (B). RT-PCR for β-actin was used as an internal control to ensure that similar concentrations of mRNA were present in all samples. For each mRNA, the first panel corresponds to positive and negative control samples prepared from indicated tissues of adult rat; the results are in agreement with several reports in the literature. The second panel shows results from E14 rat embryos, using either freshly dissected ventral spinal cord or motoneurons purified by a metrizamide-immunoaffinity method in conditions that limit de novo synthesis of mRNA. Fas and FasL are present at significant levels in motoneurons. Alternate unlabeled lanes in all panels are control in which reverse transcriptase was omitted from the incubation.
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Figure 1: Embryonic motoneurons express Fas and FasL both in situ and after purification. Total RNA was prepared from the indicated rat tissues, and RT-PCR analysis followed by Southern blotting was performed using specific primers and internal probes for FasL (A) and Fas (B). RT-PCR for β-actin was used as an internal control to ensure that similar concentrations of mRNA were present in all samples. For each mRNA, the first panel corresponds to positive and negative control samples prepared from indicated tissues of adult rat; the results are in agreement with several reports in the literature. The second panel shows results from E14 rat embryos, using either freshly dissected ventral spinal cord or motoneurons purified by a metrizamide-immunoaffinity method in conditions that limit de novo synthesis of mRNA. Fas and FasL are present at significant levels in motoneurons. Alternate unlabeled lanes in all panels are control in which reverse transcriptase was omitted from the incubation.

Mentions: We first asked whether Fas and FasL are expressed at the stage at which motoneuron PCD is about to begin. By RT-PCR followed by Southern blotting using specific oligonucleotide probes, we showed that, as reported (Suda et al. 1993; French et al. 1996), Fas and FasL were both expressed in a variety of adult tissues (Fig. 1A and Fig. B; data not shown), and that FasL was expressed by ventral spinal cord of E14 rat embryos (Fig. 1 A). In addition, we showed that Fas was expressed at significant levels by freshly isolated E14 ventral spinal cord (Fig. 1 B).


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

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

Embryonic motoneurons express Fas and FasL both in situ and after purification. Total RNA was prepared from the indicated rat tissues, and RT-PCR analysis followed by Southern blotting was performed using specific primers and internal probes for FasL (A) and Fas (B). RT-PCR for β-actin was used as an internal control to ensure that similar concentrations of mRNA were present in all samples. For each mRNA, the first panel corresponds to positive and negative control samples prepared from indicated tissues of adult rat; the results are in agreement with several reports in the literature. The second panel shows results from E14 rat embryos, using either freshly dissected ventral spinal cord or motoneurons purified by a metrizamide-immunoaffinity method in conditions that limit de novo synthesis of mRNA. Fas and FasL are present at significant levels in motoneurons. Alternate unlabeled lanes in all panels are control in which reverse transcriptase was omitted from the incubation.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Embryonic motoneurons express Fas and FasL both in situ and after purification. Total RNA was prepared from the indicated rat tissues, and RT-PCR analysis followed by Southern blotting was performed using specific primers and internal probes for FasL (A) and Fas (B). RT-PCR for β-actin was used as an internal control to ensure that similar concentrations of mRNA were present in all samples. For each mRNA, the first panel corresponds to positive and negative control samples prepared from indicated tissues of adult rat; the results are in agreement with several reports in the literature. The second panel shows results from E14 rat embryos, using either freshly dissected ventral spinal cord or motoneurons purified by a metrizamide-immunoaffinity method in conditions that limit de novo synthesis of mRNA. Fas and FasL are present at significant levels in motoneurons. Alternate unlabeled lanes in all panels are control in which reverse transcriptase was omitted from the incubation.
Mentions: We first asked whether Fas and FasL are expressed at the stage at which motoneuron PCD is about to begin. By RT-PCR followed by Southern blotting using specific oligonucleotide probes, we showed that, as reported (Suda et al. 1993; French et al. 1996), Fas and FasL were both expressed in a variety of adult tissues (Fig. 1A and Fig. B; data not shown), and that FasL was expressed by ventral spinal cord of E14 rat embryos (Fig. 1 A). In addition, we showed that Fas was expressed at significant levels by freshly isolated E14 ventral spinal cord (Fig. 1 B).

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