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

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 μM). 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.


Background
Excitotoxic neuron death has been implicated in the pathogenesis of ischemic, traumatic, and seizure-induced brain injury [1]. When glutamate receptor overactivation is intense, cell death is necrotic and characterized by a disturbance of cellular ion and volume homeostasis, leading to mitochondrial membrane potential (ΔΨm) depolarization, free radical production, ATP depletion and early plasma membrane leakage [2][3][4][5]. However, when glutamate receptor overactivation is subtle, mitochondria transiently recover their energetics, and a delayed cell death may result [3,6,7]. Under these conditions, excitotoxic neuron death is associated with the release of the proapoptotic factors cytochrome-C (cyt-C) and Apoptosis-Inducing Factor (AIF) from mitochondria [6][7][8][9][10].
The mechanisms of cyt-C and AIF release during excitotoxic neuron death remain unresolved. In the evolutionary conserved apoptosis pathway, the release of cyt-C requires the pro-apoptotic Bcl-2 family members Bax or Bak [11]. Both proteins are believed to form megachannels in the outer mitochondrial membrane large enough to release intermembrane space proteins [12]. In order to cause this increased permeability, Bax and Bak undergo a conformational change and insert into the outer mitochondrial membrane [13,14]. In non-apoptotic cells, activation of Bax or Bak is inhibited by anti-apoptotic Bcl-2 family members such as Bcl-2 and Bcl-xL. In apoptotic cells, the transcriptional induction or post-translational activation of Bcl-2-homolgy domain-3 (BH3)-only proteins overcomes this inhibition and triggers the activation of Bax and Bak and the release of cyt-C [15,16]. The release of AIF in excitotoxicity and apoptosis is likewise inhibited by Bcl-2 [10,17], suggesting that the activation of BH3-only proteins may also be required to relieve a Bcl-2 inhibition of AIF release.
Previous studies have indicated an involvement of the BH3-only protein Bad in glutamate-and Ca 2+ -induced neuronal apoptosis [18]. Interestingly, neurons from mice deficient in the BH3-only protein Bid have also been shown to be resistant to ischemic injury in vivo, as well as hypoxic and excitotoxic injury in vitro [19]. Bid is an essential component of most forms of death receptor-mediated apoptosis, and is activated post-translationally via caspase-8-mediated cleavage into a truncated form (tBid) [20,21]. tBid is subsequently myristoylated [22], triggers the activation of Bak or Bax [23,24], and induces cyt-C release from mitochondria. However, there is a growing body of evidence suggesting that caspase activation during excitotoxic neuron death may be marginal or even absent [6,8,[25][26][27]. Conversely, this suggests that Bid may trigger excitotoxic cell death through more than one pathway. Using a fluorescence resonance energy transfer single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that caspase-independent excitotoxic apoptosis induces a translocation of full length Bid (FL-Bid) from the cytosol to mitochondria [28]. In the present study, we demonstrate FL-Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons.

FL-Bid is not a significant protease substrate during excitotoxic neuron death
To investigate the involvement of apoptotic proteins in excitotoxic neuron death, we recently established a model in which a transient, 5-min exposure to the glutamate receptor agonist NMDA (300 μM) induced a delayed cell death in primary cultures of rat hippocampal neurons [7,8]. This excitotoxic cell death is characterized by mitochondrial cyt-C release and ΔΨm depolarization setting in 4 to 8 h after the NMDA exposure, followed by nuclear condensation and cell shrinkage [7,8]. We determined if FL-Bid is able to translocate to mitochondria in this model of excitotoxic apoptosis. To this end, immunoblotting experiments were performed using a rabbit polyclonal antibody (AR-53) that detects p21 FL-Bid, as well as its caspase-8-/caspase-3-generated NH 2 -terminal cleavage product [29,30]. Control experiments demonstrated that the antibody was able to detect FL-Bid and its caspase-generated NH 2 -terminal p7 fragment in HeLa D98 cells after an activation of death receptors with TNF-α/Cycloheximide(CHX) (Fig. 1A, see also Additional File 1A). The antibody was also able to detect the p7 fragment in cultured rat hippocampal neurons (Fig. 1B) exposed to the apoptosis-inducing kinase inhibitor staurosporine (STS) or TNF-α/CHX ( Fig. 1B see also Additional File 1B). However, neither a decrease in the content of FL-Bid, nor an accumulation of caspase-generated cleavage products could be detected in whole cell lysates of cultured rat hippocampal neurons exposed to NMDA (Fig. 1C, see also Additional File 1C). Activation of proteases after the NMDA exposure was however clearly detectable in the same whole cell lysates. This was evident from the decrease in the calpain I substrate full-length α-spectrin and the accumulation of calpain-specific 150 and 145 kDa α-spectrin cleavage products 4 and 8 h after the NMDA exposure (Fig. 1D).

Translocation of FL-Bid to mitochondria during excitotoxic neuron death
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).
These results were confirmed by immunofluorescence analysis of Bid redistribution during excitotoxic neuron death using the above described Bid antibody. Neurons of sham-exposed control cultures exhibited a diffuse Bid immunofluorescence signal (Fig. 2B). Cyt-C co-staining revealed a filamentous, punctate staining pattern in shamexposed controls characteristic of mitochondria. In contrast, cells that had released cyt-C in response to NMDA exhibited a clustered Bid immunofluorescence around the nucleus. Cells with released cyt C and clustered Bid immunofluorescence also exhibited nuclear chromatin condensation as evidenced by staining with the chromatin dye Hoechst 33258 (Fig. 2B).

Mild overexpression of FL-Bid or a Bid mutant that can not be cleaved by caspase-8 potently induces cell death in cultured rat hippocampal neurons
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 Death receptor mediated cleavage of Bid in HeLa cells and cultured rat hippocampal neurons Figure 1 Death receptor mediated cleavage of Bid in HeLa cells and cultured rat hippocampal neurons. Cleavage of FL-Bid in HeLa D98 cells (A)and cultured rat hippocampal neurons (B)exposed to TNF-α plus CHX (100 ng/ml plus 1 μg/ml) or staurosporine (STS, 3 μM and 300 nM, respectively) for the indicated time periods. Bid cleavage was detected by Western blot analysis. Duplicate experiments yielded similar results. (C)No detectable cleavage of FL-Bid during NMDA-induced neuronal death. Cultured rat hippocampal neurons were exposed to 300 μM NMDA in Mg 2+ -free HBS for 5 min, washed, returned to the original culture medium, and analyzed by Western blotting after 4 and 8 h. Sham-washed cultures were exposed to Mg 2+free HBS devoid of NMDA. Lack of Bid cleavage during excitotoxic neuron death was observed in four separate experiments. (D)Immunoblot analysis demonstrates significant cleavage of α-spectrin into its calpain-generated 150 and 145 kDa breakdown products. Duplicate experiments yielded similar results.
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).

Discussion
Ischemic and hypoxic injuries to the nervous system have been shown to involve the release of cell death-inducing cytokines and the activation of death receptors [19]. It is likely that these events involve the caspase-8-mediated cleavage of the BH3-only protein Bid. In support of this hypothesis, Bid-deficient animals exhibited reduced neuronal injury after cerebral ischaemia [19]. Our data provide evidence that Bid may be involved in an additional, intrinsic cell death pathway triggered by the overactivation of glutamate receptors. This pathway did not require the generation of the caspase-8-generated cleavage product tBid. Instead, we observed an efficient translocation of FL-Bid to mitochondria during excitotoxic neuron death. This translocation was associated with the mitochondrial release of pro-apoptotic factors, a process that commits cells to both caspase-dependent and caspase-independent cell death [33,34]. Although our data does not rule out the possibility that other BH3 only proteins are involved in excitotoxic apoptosis, it demonstrates that FL-Bid is sufficient to induce an apoptotic cell death in cultured rat hippocampal neurons.
In contrast to death receptor-induced apoptosis we could not detect significant amounts of cleaved Bid accumulating in the cultured rat hippocampal neurons in response to NMDA. However, we cannot exclude the possibility that Bid may be cleaved during the later stages of apoptosis, downstream of MOMP or an intracellular ion homeostasis breakdown [28,41,42]. These cells will subsequently undergo plasma membrane leakage, a proc-Translocation of FL-Bid to the mitochondria coincides with a loss of cyt-C 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 Mg 2+ -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. ess that may limit the ability to detect Bid cleavage during the late stages of apoptosis. A previous report has demonstrated that translocation of FL-Bid to mitochondria may also occur in response to an activation of death receptors when caspase-8 activation is blocked by the addition of a caspase-8 inhibitor [43]. In Bid-and caspase-8-deficient mouse embryonic fibroblasts, FL-Bid or the non-cleavable mutant FL-Bid(D59A) are also sufficient to activate apoptosis [32]. In line with this finding we found no significant difference in the ability of FL-Bid and Bid(D59A) to induce cell death in cultured rat hippocampal neurons.

FL-Bid or a Bid mutant that can not be cleaved by caspase-8 potently induces cell death in cultured rat hippocampal neurons
How may NMDA receptor overactivation stimulate FL-Bid translocation? Previous studies have demonstrated that excitotoxic neuron death is associated with a selective inhibition of phosphatidylcholine synthesis [44]. It has also been shown that physiological concentrations of phosphatidic acid and phosphatidylgycerol are able to induce an accumulation of FL-Bid in mitochondria [45]. FL-Bid has been shown to be sufficient to induce the oligomerization of Bax/Bak, resulting in its integration into the outer mitochondrial membrane triggering cyt-C release [24]. Finally, recombinant FL-Bid displayed lipid transfer activity under the same conditions and at the same nanomolar concentrations that lead to mitochondrial translocation and Cyt-C release [45]. Changes in the intracellular phospholipid environment during excitotoxic cell death signals may hence induce the translocation of FL-Bid to mitochondria and may initiate the release of pro-apoptotic factors from mitochondria.

Conclusion
Bid is highly expressed in the nervous system during embryonic and postnatal development. Interestingly and in contrast to most BH3 only proteins, Bid expression remains high in the adult nervous system [29]. The ability of both tBid and FL-Bid to translocate to mitochondria and to induce cell death suggest that this BH3-only protein is a central mediator of pathophysiological neuron death.

Generation of adenoviral vectors expressing FL-Bid or FL-Bid(D59A) in an inducible system and infection protocol
Tetracycline (tet)-on inducible adenovirus vectors expressing wild-type FL-Bid or the D59A mutant of FL-Bid that can not be cleaved by caspase-8 were generated as described previously [32]. Hippocampal neurons were infected at a MOI (multiplicity of infection) of 50 with both the FL-Bid or Bid(D59A) containing viruses and the reverse tet transactivator rtTA containing virus. One μg/ml doxycycline (Sigma) was added to the medium 24 h post infection to activate gene expression from the tet-inducible promoter [32].

Statistics
Data are given as means ± S.E.M. For statistical comparison, one-way analysis of variance followed by Tukey's test were employed. P values smaller than 0.05 were considered to be statistically significant.