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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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(a) X-gal staining of a DRG from transgenic 6-wk-old mouse progeny of the 7539 founder (progeny of the 7512 founder had similar staining pattern). (b) PCR genotyping of transgenic lines. M17Z is the original NSE-M17Z plasmid which was used to make the transgenic mouse, used here as a PCR positive control.  7512 and 7539 are positive transgenic lines.
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Figure 2: (a) X-gal staining of a DRG from transgenic 6-wk-old mouse progeny of the 7539 founder (progeny of the 7512 founder had similar staining pattern). (b) PCR genotyping of transgenic lines. M17Z is the original NSE-M17Z plasmid which was used to make the transgenic mouse, used here as a PCR positive control. 7512 and 7539 are positive transgenic lines.

Mentions: To determine whether the mutant ICE gene can also act as an inhibitor of apoptosis in vivo, and to further evaluate its mechanism of action, we established transgenic mouse lines expressing the fused mutant ICEC285GlacZ gene under the control of the NSE promoter (NSEM17Z). Transgenic mice expressing either the lacZ or bcl-2 genes under control of the NSE promoter have been well characterized, and transgene expression has been detected throughout the nervous system (22, 25, 26). PCR was used for genotyping the NSE-M17Z transgenic mice, and protein expression was detected by X-gal staining (Fig. 2). Founder mice from five different lines were crossed with C57BL/6 mice. The expression of NSE-M17Z was detected in the first and second generations of offspring, which were used in the experiments described below.


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)

(a) X-gal staining of a DRG from transgenic 6-wk-old mouse progeny of the 7539 founder (progeny of the 7512 founder had similar staining pattern). (b) PCR genotyping of transgenic lines. M17Z is the original NSE-M17Z plasmid which was used to make the transgenic mouse, used here as a PCR positive control.  7512 and 7539 are positive transgenic lines.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: (a) X-gal staining of a DRG from transgenic 6-wk-old mouse progeny of the 7539 founder (progeny of the 7512 founder had similar staining pattern). (b) PCR genotyping of transgenic lines. M17Z is the original NSE-M17Z plasmid which was used to make the transgenic mouse, used here as a PCR positive control. 7512 and 7539 are positive transgenic lines.
Mentions: To determine whether the mutant ICE gene can also act as an inhibitor of apoptosis in vivo, and to further evaluate its mechanism of action, we established transgenic mouse lines expressing the fused mutant ICEC285GlacZ gene under the control of the NSE promoter (NSEM17Z). Transgenic mice expressing either the lacZ or bcl-2 genes under control of the NSE promoter have been well characterized, and transgene expression has been detected throughout the nervous system (22, 25, 26). PCR was used for genotyping the NSE-M17Z transgenic mice, and protein expression was detected by X-gal staining (Fig. 2). Founder mice from five different lines were crossed with C57BL/6 mice. The expression of NSE-M17Z was detected in the first and second generations of offspring, which were used in the experiments described below.

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