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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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Translocation of FL-Bid to the mitochondria coincides with a loss of cyt-C. (A)Immunoblot analysis of cytosolic and mitochondria-containing pellet fractions after selective plasma membrane permeabilization in cells exposed to Mg2+-free HBS (controls) or NMDA with or without CA-074-Me. The experiment was performed in duplicate with similar results. (B)Immunofluorescence analysis of cyt-C and Bid distribution in sham- and NMDA-exposed rat hippocampal neurons. Cells were fixed 8 h after termination of the exposure. Nuclei were counterstained with Hoechst 33258. Scale bar = 5 μm.
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Figure 2: Translocation of FL-Bid to the mitochondria coincides with a loss of cyt-C. (A)Immunoblot analysis of cytosolic and mitochondria-containing pellet fractions after selective plasma membrane permeabilization in cells exposed to Mg2+-free HBS (controls) or NMDA with or without CA-074-Me. The experiment was performed in duplicate with similar results. (B)Immunofluorescence analysis of cyt-C and Bid distribution in sham- and NMDA-exposed rat hippocampal neurons. Cells were fixed 8 h after termination of the exposure. Nuclei were counterstained with Hoechst 33258. Scale bar = 5 μm.

Mentions: Next, we performed selective plasma membrane permeabilization and subsequent immunoblotting of the cytosolic fraction and the mitochondria-containing pellet fraction in cultured rat hippocampal neurons exposed to NMDA for 5 min. Immunoblotting with a voltage dependent anion carrier (VDAC) antibody demonstrated that the cytosolic fraction was not contaminated with mitochondria. Immunoblotting with the Bid antibody revealed that p21 FL-Bid translocated from the cytosol to the mitochondria-containing pellet fraction 4 h and more pronounced 8 h after termination of the NMDA exposure (Fig. 2A). This process was paralleled by the translocation of cyt-C from the mitochondria-containing pellet fraction to the cytosol (Fig. 2A) 4 h and 8 h after the NMDA exposure. Interestingly, despite a significant cyt-C decrease in the mitochondrial fraction by 8 h, the cyt-C content in the cytosolic fraction did not increase correspondingly, suggesting that cyt-C may be degraded upon release into the cytosol (see also [31]). Indeed, treatment with the membrane permeable cathepsin inhibitor CA-074 methyl ester recovered the cyt-C content in the cytosolic fraction 8 h after the NMDA exposure (Fig. 2A).


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)

Translocation of FL-Bid to the mitochondria coincides with a loss of cyt-C. (A)Immunoblot analysis of cytosolic and mitochondria-containing pellet fractions after selective plasma membrane permeabilization in cells exposed to Mg2+-free HBS (controls) or NMDA with or without CA-074-Me. The experiment was performed in duplicate with similar results. (B)Immunofluorescence analysis of cyt-C and Bid distribution in sham- and NMDA-exposed rat hippocampal neurons. Cells were fixed 8 h after termination of the exposure. Nuclei were counterstained with Hoechst 33258. Scale bar = 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1808451&req=5

Figure 2: Translocation of FL-Bid to the mitochondria coincides with a loss of cyt-C. (A)Immunoblot analysis of cytosolic and mitochondria-containing pellet fractions after selective plasma membrane permeabilization in cells exposed to Mg2+-free HBS (controls) or NMDA with or without CA-074-Me. The experiment was performed in duplicate with similar results. (B)Immunofluorescence analysis of cyt-C and Bid distribution in sham- and NMDA-exposed rat hippocampal neurons. Cells were fixed 8 h after termination of the exposure. Nuclei were counterstained with Hoechst 33258. Scale bar = 5 μm.
Mentions: Next, we performed selective plasma membrane permeabilization and subsequent immunoblotting of the cytosolic fraction and the mitochondria-containing pellet fraction in cultured rat hippocampal neurons exposed to NMDA for 5 min. Immunoblotting with a voltage dependent anion carrier (VDAC) antibody demonstrated that the cytosolic fraction was not contaminated with mitochondria. Immunoblotting with the Bid antibody revealed that p21 FL-Bid translocated from the cytosol to the mitochondria-containing pellet fraction 4 h and more pronounced 8 h after termination of the NMDA exposure (Fig. 2A). This process was paralleled by the translocation of cyt-C from the mitochondria-containing pellet fraction to the cytosol (Fig. 2A) 4 h and 8 h after the NMDA exposure. Interestingly, despite a significant cyt-C decrease in the mitochondrial fraction by 8 h, the cyt-C content in the cytosolic fraction did not increase correspondingly, suggesting that cyt-C may be degraded upon release into the cytosol (see also [31]). Indeed, treatment with the membrane permeable cathepsin inhibitor CA-074 methyl ester recovered the cyt-C content in the cytosolic fraction 8 h after the NMDA exposure (Fig. 2A).

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