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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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Whole brain lysates of NSE-M17Z mice are deficient in processing pro–IL-1β after systemic LPS administration. LPS was injected intraperitoneally (10 μg/g body weight) and 2 h before killing (wild type,  n = 4; NSE-M17Z, n = 5). PBS was injected as a control (wild type, n = 3;  NSE-M17Z, n = 6). Brains were dissected, and mature IL-1β concentration was determined using an ELISA kit specific for mature IL-1β. Results are expressed as means ± SEM.
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Figure 3: Whole brain lysates of NSE-M17Z mice are deficient in processing pro–IL-1β after systemic LPS administration. LPS was injected intraperitoneally (10 μg/g body weight) and 2 h before killing (wild type, n = 4; NSE-M17Z, n = 5). PBS was injected as a control (wild type, n = 3; NSE-M17Z, n = 6). Brains were dissected, and mature IL-1β concentration was determined using an ELISA kit specific for mature IL-1β. Results are expressed as means ± SEM.

Mentions: We next evaluated whether the mutant ICEC285G may act as a dominant negative ICE inhibitor. ICE knockout mice were almost completely defective in processing pro–IL-1β and ICE is the only protease identified so far that can process pro–IL-1β (14, 17). If the mutant ICE transgenic mice have a defect in secreting mature IL-1β, this would provide strong evidence that mutant ICEC285G can act as a dominant negative inhibitor of ICE. Systemic injection of LPS induces release of mature IL-1β, and ICE knockout mice generated by gene-targeting technique were unable to release mature IL-1β upon LPS stimulation (14, 17). To determine if our mutant ICEC285G transgenic mice are also defective in secreting mature IL-1β, we injected LPS intraperitoneally into the mutant ICEC285G transgenic mice and determined the levels of mature IL-1β in whole brain lysates using an ELISA kit which specifically detects mature IL-1β. After the systemic LPS challenge, whole brain lysates of mutant ICEC285G transgenic mice contained 74.7% less mature IL-1β as compared to that of LPS-injected wildtype mice. In control wild-type mice injected intraperitoneally with PBS, there was low but detectable levels of mature IL-1β in the brain (4.0 pg/g brain), whereas this cytokine was undetectable in the brain lysate of PBS-injected mutant ICEC285G mice (Fig. 3). Thus, mutant ICEC285G can act as an effective inhibitor of pro–IL-1β processing, strongly suggesting that mutant ICEC285G is a dominant negative inhibitor of ICE itself.


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)

Whole brain lysates of NSE-M17Z mice are deficient in processing pro–IL-1β after systemic LPS administration. LPS was injected intraperitoneally (10 μg/g body weight) and 2 h before killing (wild type,  n = 4; NSE-M17Z, n = 5). PBS was injected as a control (wild type, n = 3;  NSE-M17Z, n = 6). Brains were dissected, and mature IL-1β concentration was determined using an ELISA kit specific for mature IL-1β. Results are expressed as means ± SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196165&req=5

Figure 3: Whole brain lysates of NSE-M17Z mice are deficient in processing pro–IL-1β after systemic LPS administration. LPS was injected intraperitoneally (10 μg/g body weight) and 2 h before killing (wild type, n = 4; NSE-M17Z, n = 5). PBS was injected as a control (wild type, n = 3; NSE-M17Z, n = 6). Brains were dissected, and mature IL-1β concentration was determined using an ELISA kit specific for mature IL-1β. Results are expressed as means ± SEM.
Mentions: We next evaluated whether the mutant ICEC285G may act as a dominant negative ICE inhibitor. ICE knockout mice were almost completely defective in processing pro–IL-1β and ICE is the only protease identified so far that can process pro–IL-1β (14, 17). If the mutant ICE transgenic mice have a defect in secreting mature IL-1β, this would provide strong evidence that mutant ICEC285G can act as a dominant negative inhibitor of ICE. Systemic injection of LPS induces release of mature IL-1β, and ICE knockout mice generated by gene-targeting technique were unable to release mature IL-1β upon LPS stimulation (14, 17). To determine if our mutant ICEC285G transgenic mice are also defective in secreting mature IL-1β, we injected LPS intraperitoneally into the mutant ICEC285G transgenic mice and determined the levels of mature IL-1β in whole brain lysates using an ELISA kit which specifically detects mature IL-1β. After the systemic LPS challenge, whole brain lysates of mutant ICEC285G transgenic mice contained 74.7% less mature IL-1β as compared to that of LPS-injected wildtype mice. In control wild-type mice injected intraperitoneally with PBS, there was low but detectable levels of mature IL-1β in the brain (4.0 pg/g brain), whereas this cytokine was undetectable in the brain lysate of PBS-injected mutant ICEC285G mice (Fig. 3). Thus, mutant ICEC285G can act as an effective inhibitor of pro–IL-1β processing, strongly suggesting that mutant ICEC285G is a dominant negative inhibitor of ICE itself.

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