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Expression of a dominant negative mutant of interleukin-1 beta converting enzyme in transgenic mice prevents neuronal cell death induced by trophic factor withdrawal and ischemic brain injury.

Friedlander RM, Gagliardini V, Hara H, Fink KB, Li W, MacDonald G, Fishman MC, Greenberg AH, Moskowitz MA, Yuan J - J. Exp. Med. (1997)

Bottom Line: To explore the role of the interleukin (IL)-1 beta converting enzyme (ICE) in neuronal apoptosis, we designed a mutant ICE gene (C285G) that acts as a dominant negative ICE inhibitor.Microinjection of the mutant ICE gene into embryonal chicken dorsal root ganglial neurons inhibits trophic factor withdrawal-induced apoptosis.Our data suggest that genetic manipulation using ICE family dominant negative inhibitors can ameliorate the extent of ischemia-induced brain injury and preserve neurological function.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Charlestown 02129, USA.

ABSTRACT
To explore the role of the interleukin (IL)-1 beta converting enzyme (ICE) in neuronal apoptosis, we designed a mutant ICE gene (C285G) that acts as a dominant negative ICE inhibitor. Microinjection of the mutant ICE gene into embryonal chicken dorsal root ganglial neurons inhibits trophic factor withdrawal-induced apoptosis. Transgenic mice expressing the fused mutant ICE-lacZ gene under the control of the neuron specific enolase promoter appeared neurologically normal. These mice are deficient in processing pro-IL-1 beta, indicating that mutant ICEC285G blocks ICE function. Dorsal root ganglial neurons isolated from transgenic mice were resistant to trophic factor withdrawal-induced apoptosis. In addition, the neurons isolated from newborn ICE knockout mice are similarly resistant to trophic factor withdrawal-induced apoptosis. After permanent focal ischemia by middle cerebral artery occlusion, the mutant ICEC285G transgenic mice show significantly reduced brain injury as well as less behavioral deficits when compared to the wild-type controls. Since ICE is the only enzyme with IL-1 beta convertase activity in mice, our data indicates that the mutant ICEC285G inhibits ICE, and hence mature IL-1 beta production, and through this mechanism, at least in part, inhibits apoptosis. Our data suggest that genetic manipulation using ICE family dominant negative inhibitors can ameliorate the extent of ischemia-induced brain injury and preserve neurological function.

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Survival in vitro of NGF-dependent DRG neurons isolated  from (a) mutant ICEC285G transgenic and (b) ICE knockout newborn mice  are protected from trophic factor withdrawal–mediated apoptosis. Survival represents the percentage of neurons remaining alive after 24 h of  serum deprivation (100% at day 0). (a) The results are the average three  double blindly scored independent experiments using newborn mice  from lines 7512 and 7539. Neurons from each mouse were plated separately, and at least 400 neurons were counted per well. Results are expressed as means ± SEM. (b) Results are from an experiment double  blindly scored performed in quadruplicate, from DRG neurons isolated  from four wild-type and four ICE knockout newborn mice. At least 500  neurons were counted per well. Results are expressed as means ± SD.
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Figure 4: Survival in vitro of NGF-dependent DRG neurons isolated from (a) mutant ICEC285G transgenic and (b) ICE knockout newborn mice are protected from trophic factor withdrawal–mediated apoptosis. Survival represents the percentage of neurons remaining alive after 24 h of serum deprivation (100% at day 0). (a) The results are the average three double blindly scored independent experiments using newborn mice from lines 7512 and 7539. Neurons from each mouse were plated separately, and at least 400 neurons were counted per well. Results are expressed as means ± SEM. (b) Results are from an experiment double blindly scored performed in quadruplicate, from DRG neurons isolated from four wild-type and four ICE knockout newborn mice. At least 500 neurons were counted per well. Results are expressed as means ± SD.

Mentions: We have previously demonstrated that a CrmA-sensitive pathway, as well as ICE activation followed by endogenously produced mature IL-1β receptor binding, play important roles in trophic factor withdrawal–mediated DRG neuron apoptosis (7, 19). In this same model, we investigated whether DRG neurons isolated from mutant ICEC285G mice were resistant to cell death. DRG neurons were isolated from newborn mutant ICEC285G transgenic and wild-type mice, and cultured in the presence or absence of trophic factor. The survival of wild-type and NSE-M17Z mice DRG neurons in the presence of trophic factor were >95% after 24 h in culture. Removal of trophic factor induces 79.7% of neurons to die within 24 h. DRG neurons from two transgenic lines (7512 and 7539) were significantly protected from apoptosis in culture (48.6% cell death in 24 h) after trophic factor removal as compared to their wild-type littermates (Fig. 4 a). If resistance of DRG neurons isolated from mutant ICEC285G mice to trophic factor deprivation–induced apoptosis can be attributed to the ability of mutant ICE to inhibit endogenous ICE activity, a prediction would be that DRG neurons from ICE knockout mice should also be resistant to neuronal cell death induced by trophic factor deprivation. To test this hypothesis, we examined whether DRG neurons from ICE knockout mice are also protected from trophic factor withdrawal–mediated apoptosis. Newborn DRG neurons were isolated from mutant ICE knockout and control wild-type mice. As shown in Fig. 4 b, DRG neurons isolated from ICE knockout mice are similarly resistant to cell death induced by trophic factor deprivation as our mutant ICEC285G transgenic mice. These results suggest that mutant ICEC285G acts as a dominant negative inhibitor of ICE for the suppression of DRG neuronal cell death–induced apoptosis and that ICE plays an important role in DRG neuronal cell death induced by trophic factor deprivation.


Expression of a dominant negative mutant of interleukin-1 beta converting enzyme in transgenic mice prevents neuronal cell death induced by trophic factor withdrawal and ischemic brain injury.

Friedlander RM, Gagliardini V, Hara H, Fink KB, Li W, MacDonald G, Fishman MC, Greenberg AH, Moskowitz MA, Yuan J - J. Exp. Med. (1997)

Survival in vitro of NGF-dependent DRG neurons isolated  from (a) mutant ICEC285G transgenic and (b) ICE knockout newborn mice  are protected from trophic factor withdrawal–mediated apoptosis. Survival represents the percentage of neurons remaining alive after 24 h of  serum deprivation (100% at day 0). (a) The results are the average three  double blindly scored independent experiments using newborn mice  from lines 7512 and 7539. Neurons from each mouse were plated separately, and at least 400 neurons were counted per well. Results are expressed as means ± SEM. (b) Results are from an experiment double  blindly scored performed in quadruplicate, from DRG neurons isolated  from four wild-type and four ICE knockout newborn mice. At least 500  neurons were counted per well. Results are expressed as means ± SD.
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Related In: Results  -  Collection

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Figure 4: Survival in vitro of NGF-dependent DRG neurons isolated from (a) mutant ICEC285G transgenic and (b) ICE knockout newborn mice are protected from trophic factor withdrawal–mediated apoptosis. Survival represents the percentage of neurons remaining alive after 24 h of serum deprivation (100% at day 0). (a) The results are the average three double blindly scored independent experiments using newborn mice from lines 7512 and 7539. Neurons from each mouse were plated separately, and at least 400 neurons were counted per well. Results are expressed as means ± SEM. (b) Results are from an experiment double blindly scored performed in quadruplicate, from DRG neurons isolated from four wild-type and four ICE knockout newborn mice. At least 500 neurons were counted per well. Results are expressed as means ± SD.
Mentions: We have previously demonstrated that a CrmA-sensitive pathway, as well as ICE activation followed by endogenously produced mature IL-1β receptor binding, play important roles in trophic factor withdrawal–mediated DRG neuron apoptosis (7, 19). In this same model, we investigated whether DRG neurons isolated from mutant ICEC285G mice were resistant to cell death. DRG neurons were isolated from newborn mutant ICEC285G transgenic and wild-type mice, and cultured in the presence or absence of trophic factor. The survival of wild-type and NSE-M17Z mice DRG neurons in the presence of trophic factor were >95% after 24 h in culture. Removal of trophic factor induces 79.7% of neurons to die within 24 h. DRG neurons from two transgenic lines (7512 and 7539) were significantly protected from apoptosis in culture (48.6% cell death in 24 h) after trophic factor removal as compared to their wild-type littermates (Fig. 4 a). If resistance of DRG neurons isolated from mutant ICEC285G mice to trophic factor deprivation–induced apoptosis can be attributed to the ability of mutant ICE to inhibit endogenous ICE activity, a prediction would be that DRG neurons from ICE knockout mice should also be resistant to neuronal cell death induced by trophic factor deprivation. To test this hypothesis, we examined whether DRG neurons from ICE knockout mice are also protected from trophic factor withdrawal–mediated apoptosis. Newborn DRG neurons were isolated from mutant ICE knockout and control wild-type mice. As shown in Fig. 4 b, DRG neurons isolated from ICE knockout mice are similarly resistant to cell death induced by trophic factor deprivation as our mutant ICEC285G transgenic mice. These results suggest that mutant ICEC285G acts as a dominant negative inhibitor of ICE for the suppression of DRG neuronal cell death–induced apoptosis and that ICE plays an important role in DRG neuronal cell death induced by trophic factor deprivation.

Bottom Line: To explore the role of the interleukin (IL)-1 beta converting enzyme (ICE) in neuronal apoptosis, we designed a mutant ICE gene (C285G) that acts as a dominant negative ICE inhibitor.Microinjection of the mutant ICE gene into embryonal chicken dorsal root ganglial neurons inhibits trophic factor withdrawal-induced apoptosis.Our data suggest that genetic manipulation using ICE family dominant negative inhibitors can ameliorate the extent of ischemia-induced brain injury and preserve neurological function.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Charlestown 02129, USA.

ABSTRACT
To explore the role of the interleukin (IL)-1 beta converting enzyme (ICE) in neuronal apoptosis, we designed a mutant ICE gene (C285G) that acts as a dominant negative ICE inhibitor. Microinjection of the mutant ICE gene into embryonal chicken dorsal root ganglial neurons inhibits trophic factor withdrawal-induced apoptosis. Transgenic mice expressing the fused mutant ICE-lacZ gene under the control of the neuron specific enolase promoter appeared neurologically normal. These mice are deficient in processing pro-IL-1 beta, indicating that mutant ICEC285G blocks ICE function. Dorsal root ganglial neurons isolated from transgenic mice were resistant to trophic factor withdrawal-induced apoptosis. In addition, the neurons isolated from newborn ICE knockout mice are similarly resistant to trophic factor withdrawal-induced apoptosis. After permanent focal ischemia by middle cerebral artery occlusion, the mutant ICEC285G transgenic mice show significantly reduced brain injury as well as less behavioral deficits when compared to the wild-type controls. Since ICE is the only enzyme with IL-1 beta convertase activity in mice, our data indicates that the mutant ICEC285G inhibits ICE, and hence mature IL-1 beta production, and through this mechanism, at least in part, inhibits apoptosis. Our data suggest that genetic manipulation using ICE family dominant negative inhibitors can ameliorate the extent of ischemia-induced brain injury and preserve neurological function.

Show MeSH
Related in: MedlinePlus