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Full length Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons.

König HG, Rehm M, Gudorf D, Krajewski S, Gross A, Ward MW, Prehn JH - BMC Cell Biol. (2007)

Bottom Line: Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid) from the cytosol to mitochondria.Finally, inducible overexpression of FL-Bid or a Bid mutant that can not be cleaved by caspase-8 was sufficient to induce apoptosis in the hippocampal neuron cultures.Our data suggest that translocation of FL-Bid is sufficient for the activation of mitochondrial cell death pathways in response to glutamate receptor overactivation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and RCSI Neuroscience Research Centre, Royal College of Surgeons in Ireland, Dublin 2, Ireland. hgkoenig@rcsi.ie <hgkoenig@rcsi.ie>

ABSTRACT

Background: Bcl-2 homology domain (BH) 3-only proteins are pro-apoptotic proteins of the Bcl-2 family that couple stress signals to the mitochondrial cell death pathways. The BH3-only protein Bid can be activated in response to death receptor activation via caspase 8-mediated cleavage into a truncated protein (tBid), which subsequently translocates to mitochondria and induces the release of cytochrome-C. Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid) from the cytosol to mitochondria. We induced a delayed excitotoxic cell death in cultured rat hippocampal neurons by a 5-min exposure to the glutamate receptor agonist N-methyl-D-aspartate (NMDA; 300 microM).

Results: Western blot experiments confirmed a translocation of FL-Bid to the mitochondria during excitotoxic apoptosis that was associated with the release of cytochrome-C from mitochondria. These results were confirmed by immunofluorescence analysis of Bid translocation during excitotoxic cell death using an antibody raised against the amino acids 1-58 of mouse Bid that is not able to detect tBid. Finally, inducible overexpression of FL-Bid or a Bid mutant that can not be cleaved by caspase-8 was sufficient to induce apoptosis in the hippocampal neuron cultures.

Conclusion: Our data suggest that translocation of FL-Bid is sufficient for the activation of mitochondrial cell death pathways in response to glutamate receptor overactivation.

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FL-Bid or a Bid mutant that can not be cleaved by caspase-8 potently induces cell death in cultured rat hippocampal neurons. (A)Western blot analysis of Bid overexpression in cultured rat hippocampal neurons. Cells were co-infected with the wild-type (wt) FL-Bid adenovirus and the rtTA containing virus. Expression of Bid was induced by 1 μg/ml doxycycline treatment for 12 or 24 h. Control cells were infected but not induced. (B)Quantification of cells showing nuclear condensation in response to an overexpression of wt FL-Bid or the Bid(D59A) mutant (mt). Cells were co-infected with the wt or mt FL-Bid adenovirus and the rtTA containing virus. After 48 h, expression of Bid was induced by the addition of 1 μM doxycycline for 12 or 24 h. Cells exhibiting nuclear condensation or nuclear fragmentation were counted in 4 – 5 randomly chosen subfields after staining with the chromatin-specific dye Hoechst 33528. Data are means ± SEM from n = 3 experiments. (C)AIF immunofluorescence analysis in non-induced and induced hippocampal neurons. Cells were co-infected with the wt FL-Bid adenovirus and the rtTA containing virus and induced for 12 h. Note the mitochondrial AIF immunofluorescence in non-induced cells sparing the nuclear region, and AIF translocation to the nucleus in the induced neuron cultures. Bar = 10 μm.
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Figure 3: FL-Bid or a Bid mutant that can not be cleaved by caspase-8 potently induces cell death in cultured rat hippocampal neurons. (A)Western blot analysis of Bid overexpression in cultured rat hippocampal neurons. Cells were co-infected with the wild-type (wt) FL-Bid adenovirus and the rtTA containing virus. Expression of Bid was induced by 1 μg/ml doxycycline treatment for 12 or 24 h. Control cells were infected but not induced. (B)Quantification of cells showing nuclear condensation in response to an overexpression of wt FL-Bid or the Bid(D59A) mutant (mt). Cells were co-infected with the wt or mt FL-Bid adenovirus and the rtTA containing virus. After 48 h, expression of Bid was induced by the addition of 1 μM doxycycline for 12 or 24 h. Cells exhibiting nuclear condensation or nuclear fragmentation were counted in 4 – 5 randomly chosen subfields after staining with the chromatin-specific dye Hoechst 33528. Data are means ± SEM from n = 3 experiments. (C)AIF immunofluorescence analysis in non-induced and induced hippocampal neurons. Cells were co-infected with the wt FL-Bid adenovirus and the rtTA containing virus and induced for 12 h. Note the mitochondrial AIF immunofluorescence in non-induced cells sparing the nuclear region, and AIF translocation to the nucleus in the induced neuron cultures. Bar = 10 μm.

Mentions: We subsequently addressed the question, whether FL-Bid was sufficient to induce cell death in the hippocampal neuron cultures, and whether this cell death can occur in the absence of caspase-8-mediated Bid cleavage. Cultured rat hippocampal neurons were infected with adenoviral vectors expressing either FL-Bid or a Bid mutant that can not be cleaved by caspase-8 (D59A) under the control of a tetracycline responsive promoter [32]. Western blot analysis of cultures infected with the adenoviral vectors (50 MOI) and induced for 24 h with 1 μg/ml doxycycline demonstrated a mild overexpression of FL-Bid in the hippocampal neuron cultures (Fig. 3A). This overexpression was however sufficient to induce a massive cell death in the hippocampal neuron cultures that was characterized by cell shrinkage and nuclear condensation. Hoechst staining of nuclear chromatin revealed significant neuronal damage 24 h after induction with doxycycline, reaching a level of 80.1 ± 2.7% (Fig. 3B). FL-Bid-induced cell death was associated with the mitochondrial release of the pro-apoptotic factors cyt-C and AIF (Fig. 3C and data not shown). In contrast, hippocampal neurons that were infected with the adenoviral vectors but were not induced with doxycycline remained viable for up to 24 h, as were cells that were treated with doxycycline but not infected with the adenoviral vectors (Fig. 3B). Interestingly, overexpression of Bid(D59A) also potently induced cell death in the hippocampal neuron cultures, reaching a level of 73.5 ± 3.1% after 24 h (p > 0.1, no significant difference compared to FL-Bid induced cultures).


Full length Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons.

König HG, Rehm M, Gudorf D, Krajewski S, Gross A, Ward MW, Prehn JH - BMC Cell Biol. (2007)

FL-Bid or a Bid mutant that can not be cleaved by caspase-8 potently induces cell death in cultured rat hippocampal neurons. (A)Western blot analysis of Bid overexpression in cultured rat hippocampal neurons. Cells were co-infected with the wild-type (wt) FL-Bid adenovirus and the rtTA containing virus. Expression of Bid was induced by 1 μg/ml doxycycline treatment for 12 or 24 h. Control cells were infected but not induced. (B)Quantification of cells showing nuclear condensation in response to an overexpression of wt FL-Bid or the Bid(D59A) mutant (mt). Cells were co-infected with the wt or mt FL-Bid adenovirus and the rtTA containing virus. After 48 h, expression of Bid was induced by the addition of 1 μM doxycycline for 12 or 24 h. Cells exhibiting nuclear condensation or nuclear fragmentation were counted in 4 – 5 randomly chosen subfields after staining with the chromatin-specific dye Hoechst 33528. Data are means ± SEM from n = 3 experiments. (C)AIF immunofluorescence analysis in non-induced and induced hippocampal neurons. Cells were co-infected with the wt FL-Bid adenovirus and the rtTA containing virus and induced for 12 h. Note the mitochondrial AIF immunofluorescence in non-induced cells sparing the nuclear region, and AIF translocation to the nucleus in the induced neuron cultures. Bar = 10 μm.
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Figure 3: FL-Bid or a Bid mutant that can not be cleaved by caspase-8 potently induces cell death in cultured rat hippocampal neurons. (A)Western blot analysis of Bid overexpression in cultured rat hippocampal neurons. Cells were co-infected with the wild-type (wt) FL-Bid adenovirus and the rtTA containing virus. Expression of Bid was induced by 1 μg/ml doxycycline treatment for 12 or 24 h. Control cells were infected but not induced. (B)Quantification of cells showing nuclear condensation in response to an overexpression of wt FL-Bid or the Bid(D59A) mutant (mt). Cells were co-infected with the wt or mt FL-Bid adenovirus and the rtTA containing virus. After 48 h, expression of Bid was induced by the addition of 1 μM doxycycline for 12 or 24 h. Cells exhibiting nuclear condensation or nuclear fragmentation were counted in 4 – 5 randomly chosen subfields after staining with the chromatin-specific dye Hoechst 33528. Data are means ± SEM from n = 3 experiments. (C)AIF immunofluorescence analysis in non-induced and induced hippocampal neurons. Cells were co-infected with the wt FL-Bid adenovirus and the rtTA containing virus and induced for 12 h. Note the mitochondrial AIF immunofluorescence in non-induced cells sparing the nuclear region, and AIF translocation to the nucleus in the induced neuron cultures. Bar = 10 μm.
Mentions: We subsequently addressed the question, whether FL-Bid was sufficient to induce cell death in the hippocampal neuron cultures, and whether this cell death can occur in the absence of caspase-8-mediated Bid cleavage. Cultured rat hippocampal neurons were infected with adenoviral vectors expressing either FL-Bid or a Bid mutant that can not be cleaved by caspase-8 (D59A) under the control of a tetracycline responsive promoter [32]. Western blot analysis of cultures infected with the adenoviral vectors (50 MOI) and induced for 24 h with 1 μg/ml doxycycline demonstrated a mild overexpression of FL-Bid in the hippocampal neuron cultures (Fig. 3A). This overexpression was however sufficient to induce a massive cell death in the hippocampal neuron cultures that was characterized by cell shrinkage and nuclear condensation. Hoechst staining of nuclear chromatin revealed significant neuronal damage 24 h after induction with doxycycline, reaching a level of 80.1 ± 2.7% (Fig. 3B). FL-Bid-induced cell death was associated with the mitochondrial release of the pro-apoptotic factors cyt-C and AIF (Fig. 3C and data not shown). In contrast, hippocampal neurons that were infected with the adenoviral vectors but were not induced with doxycycline remained viable for up to 24 h, as were cells that were treated with doxycycline but not infected with the adenoviral vectors (Fig. 3B). Interestingly, overexpression of Bid(D59A) also potently induced cell death in the hippocampal neuron cultures, reaching a level of 73.5 ± 3.1% after 24 h (p > 0.1, no significant difference compared to FL-Bid induced cultures).

Bottom Line: Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid) from the cytosol to mitochondria.Finally, inducible overexpression of FL-Bid or a Bid mutant that can not be cleaved by caspase-8 was sufficient to induce apoptosis in the hippocampal neuron cultures.Our data suggest that translocation of FL-Bid is sufficient for the activation of mitochondrial cell death pathways in response to glutamate receptor overactivation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and RCSI Neuroscience Research Centre, Royal College of Surgeons in Ireland, Dublin 2, Ireland. hgkoenig@rcsi.ie <hgkoenig@rcsi.ie>

ABSTRACT

Background: Bcl-2 homology domain (BH) 3-only proteins are pro-apoptotic proteins of the Bcl-2 family that couple stress signals to the mitochondrial cell death pathways. The BH3-only protein Bid can be activated in response to death receptor activation via caspase 8-mediated cleavage into a truncated protein (tBid), which subsequently translocates to mitochondria and induces the release of cytochrome-C. Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid) from the cytosol to mitochondria. We induced a delayed excitotoxic cell death in cultured rat hippocampal neurons by a 5-min exposure to the glutamate receptor agonist N-methyl-D-aspartate (NMDA; 300 microM).

Results: Western blot experiments confirmed a translocation of FL-Bid to the mitochondria during excitotoxic apoptosis that was associated with the release of cytochrome-C from mitochondria. These results were confirmed by immunofluorescence analysis of Bid translocation during excitotoxic cell death using an antibody raised against the amino acids 1-58 of mouse Bid that is not able to detect tBid. Finally, inducible overexpression of FL-Bid or a Bid mutant that can not be cleaved by caspase-8 was sufficient to induce apoptosis in the hippocampal neuron cultures.

Conclusion: Our data suggest that translocation of FL-Bid is sufficient for the activation of mitochondrial cell death pathways in response to glutamate receptor overactivation.

Show MeSH
Related in: MedlinePlus