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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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Microinjection of β-actin-M17Z protects embryonic chicken  DRG neurons from trophic factor withdrawal–induced apoptosis. Survival represents the percentage of injected E10 chick DRG neurons remaining alive at 3 and 6 d (100% at day 0) after trophic factor deprivation. A  total of 783 neurons were injected with vector alone (solid bar), 421 with  dye alone (empty bar), and 850 with β-actin-M17Z (hatched bar). Each  condition was done on at least four independent experiments. Results are  expressed as means ± SEM.
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Figure 1: Microinjection of β-actin-M17Z protects embryonic chicken DRG neurons from trophic factor withdrawal–induced apoptosis. Survival represents the percentage of injected E10 chick DRG neurons remaining alive at 3 and 6 d (100% at day 0) after trophic factor deprivation. A total of 783 neurons were injected with vector alone (solid bar), 421 with dye alone (empty bar), and 850 with β-actin-M17Z (hatched bar). Each condition was done on at least four independent experiments. Results are expressed as means ± SEM.

Mentions: Survival of DRG neurons in culture requires the presence of trophic factors which include nerve growth factor and serum. In the absence of trophic factor support, DRG neurons undergo apoptosis (24). To determine whether the mutant ICE can inhibit DRG neuronal death induced by trophic factor deprivation, primary cultures of chicken embryonic DRG neurons were microinjected with a construct of the fused mutant ICEC285G-lacZ gene under the control of the β-actin promoter (β-actinM17Z). Neurons were co-injected with rhodamine-isothiocyanate dextran as a marker and with Hoechst dye to determine neuronal nuclear morphology. After trophic factor removal, control neurons microinjected with the β-actinlacZ construct survived 22.5 and 6.0% after 3 and 6 d in culture, respectively. No significant difference was detected when compared to cells injected with dye alone. In contrast, neurons injected with β-actin-M17Z survived 85.0 and 81.0% after 3 and 6 d in culture, respectively (Fig. 1). These results showed that the mutant ICE gene inhibits DRG neuronal cell death induced by trophic factor deprivation, suggesting that mutant ICE may be able to suppress the activities of wild-type ICE or ICE-like proteases.


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)

Microinjection of β-actin-M17Z protects embryonic chicken  DRG neurons from trophic factor withdrawal–induced apoptosis. Survival represents the percentage of injected E10 chick DRG neurons remaining alive at 3 and 6 d (100% at day 0) after trophic factor deprivation. A  total of 783 neurons were injected with vector alone (solid bar), 421 with  dye alone (empty bar), and 850 with β-actin-M17Z (hatched bar). Each  condition was done on at least four independent experiments. Results are  expressed as means ± SEM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196165&req=5

Figure 1: Microinjection of β-actin-M17Z protects embryonic chicken DRG neurons from trophic factor withdrawal–induced apoptosis. Survival represents the percentage of injected E10 chick DRG neurons remaining alive at 3 and 6 d (100% at day 0) after trophic factor deprivation. A total of 783 neurons were injected with vector alone (solid bar), 421 with dye alone (empty bar), and 850 with β-actin-M17Z (hatched bar). Each condition was done on at least four independent experiments. Results are expressed as means ± SEM.
Mentions: Survival of DRG neurons in culture requires the presence of trophic factors which include nerve growth factor and serum. In the absence of trophic factor support, DRG neurons undergo apoptosis (24). To determine whether the mutant ICE can inhibit DRG neuronal death induced by trophic factor deprivation, primary cultures of chicken embryonic DRG neurons were microinjected with a construct of the fused mutant ICEC285G-lacZ gene under the control of the β-actin promoter (β-actinM17Z). Neurons were co-injected with rhodamine-isothiocyanate dextran as a marker and with Hoechst dye to determine neuronal nuclear morphology. After trophic factor removal, control neurons microinjected with the β-actinlacZ construct survived 22.5 and 6.0% after 3 and 6 d in culture, respectively. No significant difference was detected when compared to cells injected with dye alone. In contrast, neurons injected with β-actin-M17Z survived 85.0 and 81.0% after 3 and 6 d in culture, respectively (Fig. 1). These results showed that the mutant ICE gene inhibits DRG neuronal cell death induced by trophic factor deprivation, suggesting that mutant ICE may be able to suppress the activities of wild-type ICE or ICE-like proteases.

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