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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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Protection from permanent MCA occlusion–mediated infarct  in NSE-M17Z (black) compared to wild-type (white) mice. (A) Neurological grading 30 min and 24 h after occlusion. Neurological grading: 0, no  neurological deficits; 1, failure to extend the right forepaw; 2, circling to  the contralateral side; 3, loss of walking or righting reflex. (B) Infarct area  assessed at 24 h. (C) Regional cerebral blood flow (rCBF), and mean arterial blood pressure (MBP) of wild-type and transgenic mice during 30 min  of ischemia (**, P <0.01).
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Figure 5: Protection from permanent MCA occlusion–mediated infarct in NSE-M17Z (black) compared to wild-type (white) mice. (A) Neurological grading 30 min and 24 h after occlusion. Neurological grading: 0, no neurological deficits; 1, failure to extend the right forepaw; 2, circling to the contralateral side; 3, loss of walking or righting reflex. (B) Infarct area assessed at 24 h. (C) Regional cerebral blood flow (rCBF), and mean arterial blood pressure (MBP) of wild-type and transgenic mice during 30 min of ischemia (**, P <0.01).

Mentions: To determine if ICE plays a role in apoptosis induced by ischemic insult and if the mutant ICEC285G may act to reduce ischemic brain injury, we investigated whether the mutant ICEC285G transgenic mice were protected in a mouse focal cerebral ischemia model where apoptotic cell death has been reported (27). We performed permanent MCA occlusion in 14 wild-type and 11 transgenic mice, progeny of five different founder mice (7509, 7512, 7516, 7538, and 7539). Mice were scored neurologically 30 min and 24 h after the occlusion. In the initial 30 min evaluation, there was no significant difference in the neurological score. During the ensuing 24 h, however, wild-type mice remained impaired, while transgenic mice improved neurologically (Fig. 5 A). Mice were killed at 24 h, and infarct volume was quantified with 4% 2,3,5-triphenyltetrazolium chloride. Infarct volume was significantly smaller in NSE-M17Z (66 ± 11 mm3 [n = 11]) when compared to the wild-type littermate mice (125 ± 5 mm3 [n = 14]; Fig. 5 B). Physiologic parameters were recorded in a separate set of transgenic and wild-type mice. Blood pressure, arterial blood gases (PO2, PCO2, and blood pH), regional cerebral blood flow, and body temperature did not significantly differ in the two sets of mice, before and throughout 30 min of ischemia (Fig. 5 C). Thus, expression of mutant ICEC285G, a dominant negative inhibitor of ICE, protects neurons from ischemic insult.


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)

Protection from permanent MCA occlusion–mediated infarct  in NSE-M17Z (black) compared to wild-type (white) mice. (A) Neurological grading 30 min and 24 h after occlusion. Neurological grading: 0, no  neurological deficits; 1, failure to extend the right forepaw; 2, circling to  the contralateral side; 3, loss of walking or righting reflex. (B) Infarct area  assessed at 24 h. (C) Regional cerebral blood flow (rCBF), and mean arterial blood pressure (MBP) of wild-type and transgenic mice during 30 min  of ischemia (**, P <0.01).
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Related In: Results  -  Collection

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Figure 5: Protection from permanent MCA occlusion–mediated infarct in NSE-M17Z (black) compared to wild-type (white) mice. (A) Neurological grading 30 min and 24 h after occlusion. Neurological grading: 0, no neurological deficits; 1, failure to extend the right forepaw; 2, circling to the contralateral side; 3, loss of walking or righting reflex. (B) Infarct area assessed at 24 h. (C) Regional cerebral blood flow (rCBF), and mean arterial blood pressure (MBP) of wild-type and transgenic mice during 30 min of ischemia (**, P <0.01).
Mentions: To determine if ICE plays a role in apoptosis induced by ischemic insult and if the mutant ICEC285G may act to reduce ischemic brain injury, we investigated whether the mutant ICEC285G transgenic mice were protected in a mouse focal cerebral ischemia model where apoptotic cell death has been reported (27). We performed permanent MCA occlusion in 14 wild-type and 11 transgenic mice, progeny of five different founder mice (7509, 7512, 7516, 7538, and 7539). Mice were scored neurologically 30 min and 24 h after the occlusion. In the initial 30 min evaluation, there was no significant difference in the neurological score. During the ensuing 24 h, however, wild-type mice remained impaired, while transgenic mice improved neurologically (Fig. 5 A). Mice were killed at 24 h, and infarct volume was quantified with 4% 2,3,5-triphenyltetrazolium chloride. Infarct volume was significantly smaller in NSE-M17Z (66 ± 11 mm3 [n = 11]) when compared to the wild-type littermate mice (125 ± 5 mm3 [n = 14]; Fig. 5 B). Physiologic parameters were recorded in a separate set of transgenic and wild-type mice. Blood pressure, arterial blood gases (PO2, PCO2, and blood pH), regional cerebral blood flow, and body temperature did not significantly differ in the two sets of mice, before and throughout 30 min of ischemia (Fig. 5 C). Thus, expression of mutant ICEC285G, a dominant negative inhibitor of ICE, protects neurons from ischemic insult.

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