death executioner Bcl-2 homologue
Since Debcl induces cell death, which is partly inhibited by the overexpression of Bcl-2 and Bcl-xL, an attractive hypothesis is that Debcl binds to and neutralizes prosurvival Bcl-2 homologs. Because no prosurvival Bcl-2–like proteins have been identified so far in Drosophila, a test was performed to see if Debcl can bind to any of the known mammalian or viral prosurvival homologs of Bcl-2. In transient overexpression experiments in mammalian cells, Debcl associates with Bcl-2 and most of its functional homologs, although the binding to Bcl-xL, Mcl-1, and adenovirus E1B19K protein is relatively weaker. For these interaction studies, the method involved radiolabeled cell extracts that allow the simultaneous detection of two interacting proteins in the same sample. Conventional immunoblotting of the immunoprecipitated proteins was also used and similar results were obtained. These data clearly show that Debcl can interact with most of the known prosurvival Bcl-2 proteins and is likely to induce cell death by the same molecular mechanisms as other proapoptotic Bcl-2-related proteins. These results further provide evidence for the functional conservation of Bax-like proteins in mammals and flies. A test was also performed to see whether Debcl interacts with the proapoptotic members of Bcl-2 family. In coimmunoprecipitation experiments, Debcl does not associate with any of the BH3-only proteins (Bik, Bid, Bad, and Bim) or the BH1-, BH2-, and BH3- containing proteins (Bax and Bak) (Colussi, 2000).
Bcl-2 family proteins are key regulators of apoptosis. Both pro-apoptotic and anti-apoptotic members of this family are found in mammalian cells, but only the pro-apoptotic protein Debcl has been characterized in Drosophila. Buffy, the second Drosophila Bcl-2-like protein, is a pro-survival protein. Ablation of Buffy by RNA interference leads to ectopic apoptosis, whereas overexpression of buffy results in the inhibition of developmental programmed cell death and gamma irradiation-induced apoptosis. Buffy interacts genetically and physically with Debcl to suppress Debcl-induced cell death. Genetic interactions suggest that Buffy acts downstream of Reaper, Grim and Head involution defective, and upstream of the apical caspase Dronc. Furthermore, overexpression of buffy inhibits ectopic cell death in diap1 (th5) mutants. Taken together these data suggest that Buffy can act downstream of Rpr, Grim and Hid to block caspase-dependent cell death. Overexpression of Buffy in the embryo results in inhibition of the cell cycle, consistent with a G1/early-S phase arrest. These data suggest that Buffy is functionally similar to the mammalian pro-survival Bcl-2 family of proteins (Quinn, 2003).
The mammalian pro-apoptotic Bcl-2 proteins function by binding and sequestering pro-survival Bcl-2 members. Debcl binds most mammalian pro-survival Bcl-2 proteins, including Bcl-2 and Bcl-XL, but not their pro-apoptotic counterparts. In order to determine whether Debcl heterodimerizes with Buffy, co-immunoprecipitation experiments were carried out. FLAG-tagged Buffy was coexpressed with HA-tagged Debcl in 293T cells. Immunoprecipitation was performed with anti-FLAG or anti-HA antibodies. The control immunoblot with anti-FLAG shows that FLAG-Buffy (33 kDa) is precipitated. Immunoblotting of the FLAG immunoprecipitates with anti-HA reveals the HA-Debcl protein, suggesting that the two proteins can co-immunoprecipitate. Therefore, like the pro- and anti-apoptotic members of the mammalian Bcl-2 family, Debcl and Buffy can physically interact (Quinn, 2003).
A Drosophila homolog of the human killer protein Bok (termed in this study DBok but this Interactive Fly report will conform to the FlyBase term Debcl) was identified. The predicted structure of Debcl is similar to pore-forming Bcl-2/Bax family members. Debcl induces apoptosis in insect and human cells, which is suppressible by anti-apoptotic human Bcl-2 family proteins. A caspase inhibitor suppresses Debcl-induced apoptosis but does not prevent Debcl-induced cell death. Moreover, Debcl targets mitochondria and triggers cytochrome c release through a caspase-independent mechanism. These characteristics of Debcl reveal evolutionary conservation of cell death mechanisms in flies and humans (Zhang, 2000).
Using the human Bcl-2 sequence for data base searches, an EST clone (AI513093) was identified and cDNAs were subsequently cloned encoding a 214-amino acid protein, which contains regions sharing extensive amino acid sequence homology with the BH1, BH2, and BH3 domains of other Bcl-2 family proteins. Overall, the sequence of this protein is most similar to rat Bok, a pro-apoptotic Bcl-2 family member, with 30% sequence identity (52% similarity). The homology of Debcl to other Bcl-2 family proteins was further analyzed by modeling the protein on the structure of human Bcl-XL, confirming the prediction that Debcl adopts a highly similar fold of an irregular alpha-helical bundle, resembling the pore-forming domains of bacterial toxins. In particular, six alpha-helices are predicted, including a hairpin pair of largely hydrophobic alpha-helices in the center of the molecule, surrounded by a shell of four amphipathic alpha-helices (Zhang, 2000).
To explore its effects on apoptosis, the Debcl protein was overexpressed by transient transfection in Sf9 insect cells, along with a GFP marker. Alternatively, Debcl was expressed as a GFP fusion protein, providing a convenient means of verifying expression. Staining of the fixed cells with a DNA-binding fluorochrome (DAPI) 1 day later revealed the presence of multiple apoptotic cells (with condensed chromatin, fragmented nuclei, and rounded, shrunken cell bodies) in cultures of Debcl- but not control plasmid-transfected Sf9 cells. Debcl also induces apoptosis of mammalian cells, such as HEK293, HT1080, and COS-7, implying evolutionary conservation of its cytotoxic function. Debcl-induced apoptosis is consistently suppressed by co-expressing the anti-apoptotic Bcl-2 family protein Mcl-1. Immunoblot analysis confirms that Debcl protein is still produced, and indeed is present at higher levels when cells are co-transfected with Mcl-1, suggesting that the cytoprotective effects of this anti-apoptotic Bcl-2 family protein allows the Debcl protein to accumulate to higher levels. Bcl-XL also partially inhibits Debcl-induced apoptosis but is far more variable in its effects, inhibiting Debcl-induced apoptosis by 0%-50% (mean = 19%). The BH3 domain of many pro-apoptotic Bcl-2 family proteins is required for dimerization with anti-apoptotic proteins such as Bcl-2 and Bcl-XL and for induction of cell death. However, similar to mammalian Bok, deletion of the BH3 domain of Debcl does not interfere with apoptosis induction by Debcl, indicating a BH3-independent mechanism (Zhang, 2000).
Based on fluorescence UV microscopy analysis of cells transfected with a plasmid producing GFP-Debcl fusion protein, the Debcl protein was determined to be cytosolic, localizing to intracellular organelles in a pattern typical of Bcl-2 family proteins. Subcellular fractionation studies confirm that Debcl is associated at least in part with mitochondria-containing heavy-membrane fractions, despite the absence of the C-terminal transmembrane domain, which is commonly found in many other Bcl-2 family proteins (Zhang, 2000).
The mammalian pro-apoptotic Bcl-2 family members, which share structural similarity with pore-forming proteins (Bax, Bak, and Bid), induce release of cytochrome c from mitochondria. Similarly, in transfection experiments, expression of Debcl in 293T cells induces an increase in cytosolic and a concomitant decrease in mitochondrial cytochrome c. Moreover, similar to mammalian Bax, release of cytochrome c is caspase-independent, as evidenced by failure of a broad spectrum caspase inhibitor, zVAD-fmk, to suppress it. Under the same conditions, however, zVAD-fmk suppresses Debcl-induced caspase activation (determined by measuring cleavage of the caspase substrate acetyl-Asp-Glu-Val-Asp-aminomethyl-coumarin (Ac-DEVD-AFC) and apoptosis, as determined by DAPI staining of fixed cells. Thus, zVAD-fmk effectively blocks the caspase activation that occurs downstream of cytochrome c release in Debcl-expressing cells. However, analogous to previous reports for mammalian Bax, Debcl-induces non-apoptotic cell death (as determined by failure of cells to exclude trypan blue dye) in the presence of caspase inhibitor, presumably due to the deleterious consequences of cytochrome c release on mitochondrial metabolic function. In contrast to zVAD-fmk, anti-apoptotic Mcl-1 protein suppresses Debcl-induced cytochrome c release, as well as Debcl-induced apoptosis and cell death. Taken together, these findings suggest that Debcl induces cell death through mechanisms similar to those employed by its mammalian counterparts (Zhang, 2000).
To determine whether the C-terminal hydrophobic region of Rob-1 (AKA Debcl) is necessary for its intracytoplasmic membrane docking or proapoptotic function, an expression plasmid producing a truncated form of Rob-1 (Drob-deltaTM) lacking the last 39-aa residues, which contain the putative transmembrane domain, was constructed. In contrast to the membrane localization of full-length Rob-1, the Drob-deltaTM mutant displays a diffuse distribution throughout the cytosol, when overexpressed in either COS7 cells or S2 cells. A cell death assay revealed that this mutation dramatically reduces the ability of Rob-1 to kill S2 cells. Furthermore, the caspase-stimulating activity of the Drob-deltaTM mutant is completely eliminated. These results suggest that the cell killing and caspase-stimulating activities of Rob-1 both require that it be targeted to subcellular components, most likely to the mitochondria, through its C-terminal transmembrane domain (Igaki, 2000).
See the embryonic expression pattern of debcl at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
Because of the low expression of debcl, tyramide amplification was used after hybridization to detect Debcl mRNA expression in situ. In early embryos, Debcl mRNA is present uniformly, but becomes more concentrated in the tissues of the gut later in embryogenesis. The relatively high levels of Debcl RNA in early embryos are likely to be derived maternally, since zygotic transcription does not begin until stage 5. From stage 14 embryos, Debcl mRNA is detected in regions in the head that correspond to the pharynx and clypeolabrum, where many TUNEL positive cells (cells undergoing apoptosis as reflected in DNA fragmentation) are detected. Expression can also be detected in a segmentally reiterated pattern in stage 14 embryos that may correlate with the TUNEL positive cells that are detected in the central nervous system at this stage. During 3rd instar larval development, Debcl expression is detected in the brain lobes in the outer proliferative center, in the posterior part of the eye imaginal disc, and in the gut, where TUNEL positive cells are clearly seen. Debcl expression is also clearly evident in the salivary glands, particularly in the ducts. Because of background staining problems in salivary glands, acridine orange staining instead of TUNEL was used to detect apoptotic cells in this tissue. Using this technique, no apoptotic cells were detected in 3rd instar salivary glands, suggesting that Debcl expression may precede cell death in this tissue. High levels of Debcl expression are detected in the nurse cell compartment of stage 10a ovaries, which undergo apoptosis at stage 10b. Thus, Debcl expression late in embryogenesis, during larval development, and during oogenesis significantly correlates with tissues undergoing apoptosis (Colussi, 2000).
Northern blot analysis of Rob-1 has detected a 1.3-kb transcript, consistent with the size of the cDNA. RT-PCR analysis demonstrates that Rob-1 mRNA is expressed at all stages of development. In addition, in situ hybridization with digoxigenin-labeled Rob-1 RNA probes reveals that Rob-1 is highly expressed in early-stage embryos. As development proceeds, expression levels of Rob-1 mRNA are reduced. In the third-instar larval stages, Rob-1 is expressed in leg discs, eye discs, wing discs, and midgut, but no signal was detected in salivary glands and brain (Igaki, 2000).
To determine subcellular localization, HA-tagged Rob-1 was overexpressed in S2 cells, and the cells were stained with an anti-HA antibody. Confocal microscopy reveals that Rob-1 has a granular cytoplasmic distribution, consistent with an association with intracellular organelles. To better identify the localization of Rob-1, transfected cells were colabeled with a mitochondria-specific fluorescent dye, MitoTracker. The pattern of mitochondrial staining is similar to that of Rob-1 . Merged images of Rob-1- and MitoTracker-stained cells show that the two proteins colocalize. This colocalization is also observed when HA-Rob-1 is overexpressed in mammalian COS7 cells. Subcellular fractionation studies in S2 cells reveals that HA-Rob-1 is enriched in the heavy-membrane fraction. HA-Rob-1 is also present in the low speed pellet and light membrane fractions with a smaller amount of staining but not in the cytosolic fraction. Together with the microscopic data, these results suggest that Rob-1 localizes to the intracytoplasmic membranes, predominantly to the mitochondrial membranes (Igaki, 2000).
After screening a number of UAS-debcl lines, two lines (UAS-debcl#26 on chromosome III and UAS-debcl#18 on chromosome II) were found that, when crossed to GMR-GAL4, give rise to adults with severely ablated eyes. To use this phenotype to examine genetic interactions, a stock was generated containing GMR-GAL4 (2nd chromosome) and UAS-debcl#26. To examine whether the rough eye phenotype is due to the activity of caspases, GMR-p35 was crossed to these flies and the eye phenotype of the progeny was examined. GMR-p35 significantly improves the severe rough eye phenotype of GMR-GAL4; UAS-debcl#26 eyes. These results confirm that Debcl functions in a caspase-dependent fashion upstream of caspase activation (Colussi, 2000).
To determine the involvement of rpr, hid, or grim in the GMR-GAL4; UAS-debcl#26 eye phenotype, these flies were crossed to a deficiency that removes all three genes (Df(3L)H99). If rpr, hid, or grim are rate limiting for Debcl function, then suppression of the GMR-GAL4; UAS-debcl#26 eye phenotype would be expected. However, no significant suppression of this phenotype was observed, suggesting that the GMR-GAL4; UAS-debcl#26 eye phenotype is not dependent on the gene dosage of rpr, hid, or grim (Colussi, 2000).
Next, a test was performed to see whether the inhibitor of apoptosis (IAP) homolog, diap1, genetically interacts with debcl, by examining the GMR-GAL4; UAS-debcl#26 eye phenotype when the dosage of diap1 is halved. Halving the dosage of diap1, using two different deficiencies, results in a strong enhancement of the GMR-GAL4; UAS-debcl#26 eye phenotype. Furthermore, there is a significant reduction in the number of flies expected to contain either of the diap1 deficiencies and GMR-GAL4; UAS-debcl#26. This is possibly due to leaky expression of the GMR-GAL4; UAS-debcl#26 construct in other tissues during development and the enhancement of this effect by reducing the dose of diap1. Thus, diap1 genetically interacts with debcl. No genetic interaction between debcl and diap2 was observed when a diap2 deficiency was crossed with GMR-GAL4; UAS-debcl#26 (Colussi, 2000).
Described recently has been a mutation in the Drosophila Apaf1/ced4 homolog dark, (Rodriguez, 1999). To assess the effect of reducing the dosage of dark on the GMR-GAL4; UAS-debcl#26 eye phenotype, a hypomorphic allele of dark (darkCD8) was crossed to GMR-GAL4/CyO; UAS-debcl#26/TM6B flies. Reducing the dosage of dark suppresses the rough eye phenotype of GMR-GAL4; UAS-debcl#26 flies. Therefore, dark genetically interacts with debcl (Colussi, 2000).
Currently, no specific debcl mutants are available. Therefore, to examine the in vivo function of Debcl, RNAi studies were carried out to inhibit debcl gene function. RNAi is a technique whose effects mimic mutation; it is a powerful technique used to disrupt the function of specific genes. Additionally, RNAi has the advantage of ablating maternally contributed mRNA; this is difficult to achieve genetically. Precellularized embryos were injected with debcl double-stranded RNA, aged until stage 16, and analyzed by TUNEL staining, in order to detect apoptotic cells. debcl RNAi results in a large reduction in TUNEL positive cells in the embryos. These RNAi results indicate that debcl gene function is required for programed cell death in the embryos. Additionally, these data suggest that maternally deposited debcl mRNA may be required for normal cell death in fly embryos (Colussi, 2000).
Removal of Debcl function during Drosophila embryonic development results in excess glial cells, demonstrating its pro-apoptotic function. In cell culture assays, Debcl efficiently induces apoptosis but, remarkably, also demonstrates protective activity when death stimuli are introduced. Ectopic expression of Debcl in the eye leads to subtle defects that are strongly potentiated by ultraviolet irradiation, resulting in a dramatic loss of retinal cells (Brachmann, 2000).
Two Bcl-2 family members, Debcl and dBorg-2, have been identified in Drosophila. Both proteins contain BH1, BH2 and BH3 domains and are most similar to Bok/Mtd, a pro- apoptotic family member. Apart from an amino-terminal extension in Debcl, residues conserved within the Bcl-2 family are present throughout the length of the sequences and are concentrated in the BH domains. Both debcl and dborg-2 are expressed between embryonic stages 11 and 16, stages of maximal cell death and dynamically throughout development of the fly. These results suggest a role in developmentally regulated cell death; however, the overall pattern of transcription does not distinguish between a pro- or anti-apoptotic function for these molecules. A series of cell culture assays were undertaken to investigate both possibilities. Overexpression of debcl promotes both death and survival in cell culture assays. Overexpression of debcl in BHK.21, HEK 293, Chinese hamster ovary (CHO) and Drosophila Schneider S2 cells resulted in reduced viability, indicating a strong conservation of killing function across species. A dose-response curve of the killing effect of Debcl in CHO cells demonstrates the potency of this pro-apoptotic Bcl-2 family member: when 1 µg of the debcl expression vector is used, 9% of the transfected cells are alive 48 hours later. Decreasing the amount of debcl expression vector to 50 ng increases the survival rate to only 50% (Brachmann, 2000).
Co-expression of the baculovirus caspase inhibitor p35 and debcl in CHO cells indicates that the majority of cell killing by Debcl is through a caspase-dependent mechanism. Surprisingly, Schneider S2 cells are relatively resistant to killing by debcl expression, since transfections with less than 0.5 µg of debcl expression vector does not appreciably kill S2 cells. COS cells are also refractory to killing by Debcl. Perhaps these two cell types contain high levels of a protective Bcl-2 family member or do not normally express activators or cofactors required for the pro-apoptotic activity of Debcl (Brachmann, 2000).
Since there is sequence similarity between Debcl and the anti-apoptotic protein Bcl-2, the possibility that Debcl might rescue cells from death was examined. Following transfection of Drosophila S2 cells with an amount of debcl expression plasmid that results in no appreciable cell killing, apoptosis was induced in two ways: (1) by expressing the C. elegans caspase CED-3 and (2) by serum deprivation. In both cases, a significant increase in the number of viable cells after 24 hours is consistently observed, demonstrating that this pro-apoptotic protein can also act as a pro-survival molecule. Such a functional conversion of a Bcl-2 family member has been described previously -- Bcl-2 becomes a killer molecule upon cleavage. Similarly, it is possible that a post-translational modification could turn Debcl into a protective molecule. Alternatively, overexpression of debcl might lead to the up-regulation of a protective pathway in S2 cells, resulting in resistance to apoptosis (Brachmann, 2000).
RNA-mediated interference of debcl results in excess glial cells. An RNA interference assay (RNAi) was used to mimic loss of debcl function and to inactivate the maternal contribution of debcl mRNA. Double-stranded debcl RNA (dsRNA) was injected into the posterior pole region of pre-cellular blastoderm embryos. TUNEL analysis of stage 12/13 embryos injected with debcl dsRNA shows a reduction in TUNEL-positive cells, as compared with a wild-type embryo, indicating that developmental PCD is inhibited. To further assess the development of embryos injected with debcl dsRNA, stage 16 embryos were examined with an antibody to a glial-specific marker. A dramatic expansion of glial cells into the posterior region is observed near the site of injection in 44% of the debcl dsRNA-injected embryos. This phenotype is never observed in control embryos injected with white dsRNA. Thus, reduction of Debcl activity leads to ectopic cells at the site of injection, including a large number of glia, suggesting that this excess tissue is contiguous with the central nervous system (CNS). Fusion of the most posterior embryonic segments, A8 and A9, requires PCD and aids contraction of the CNS. Inhibition of PCD may have resulted in A8 and A9 remaining distinct, leading to an extended CNS region wrapped around excess posterior tissue. Alternatively, excess glial cells may be a response to injection trauma and Debcl might be required not only for normal developmental apoptosis, but also to remove these glial cells (Brachmann, 2000).
To further assess the in vivo role of Debcl, GMR-debcl transgenic flies were created that express debcl exclusively in the developing retina. None of the independent transgenic lines exhibits the expected ablated eye phenotype seen upon overexpression of other death-inducers such as reaper or grim; however, occasional interommatidial pigment cells are missing and bristle groups are mispositioned, suggesting a failure in the correct regulation of interommatidial cell position or removal (Brachmann, 2000).
The subtle phenotype of the GMR-debcl flies suggests that simple overexpression of debcl is insufficient to kill and raises the possibility that Debcl requires appropriate activation before inducing apoptosis in vivo, an idea consistent with the broad expression of debcl in embryonic tissues. One candidate activator is the DNA damage response pathway. UV irradiation was used to determine the ability of DNA damage to potentiate Debcl function. High levels of irradiation (50,000 µJ/cm2)result in a nearly ablated eye. Lower doses (20,000 µJ/cm2) result in only minor loss of cells. Concomitant expression of debcl results in dramatically enhanced cell killing and a severely reduced eye; this phenotype is partially rescued by co-expression of the baculovirus protein p35, an inhibitor of caspase function. These results indicate a strong synergy between death induced by Debcl and the DNA damage response pathway. The potentiation of UV-irradiation-induced death by ectopically expressed debcl suggests that the killer function of Debcl is post-transcriptionally regulated. Previous work in cell culture has led to the idea that the balance of pro-and anti-apoptotic Bcl-2 family members is critical for determining whether a cell is permitted to survive. Therefore, a vast excess of an as yet unknown, anti-apoptotic Bcl-2 family member in the developing eye could, by hetero-dimerization, neutralize the Debcl pro-apoptotic function (as observed for Bax with Bcl-2). Alternatively, Debcl may require additional post-translational modifications to be fully activated, modifications that are not provided during the normal development of the fly eye. By analogy with known Bcl-2 family members, this could be through conformational change (as observed for Bax), cleavage (as described for Bcl-2) or by phosphorylation (as is the case for Bad) (Brachmann, 2000 and references therein).
Bcl-2 family proteins regulate cell death through the mitochondrial apoptotic pathway. Here, we show that the Drosophila Bax-like Bcl-2 family protein Drob-1 (Debcl) maintains mitochondrial function to protect cells from neurodegeneration. A pan-neuronal knockdown of Drob-1 results in lower locomotor activity and a shorter lifespan in adult flies. Either the RNAi-mediated downregulation of Drob-1 or overexpression of Drob-1 antagonist Buffy strongly enhances the polyglutamine-induced accumulation of ubiquitinated proteins and subsequent neurodegeneration. Furthermore, ectopic expression of Drob-1 suppresses the neurodegeneration and premature death of flies caused by expanded polyglutamine. Drob-1 knockdown decreases cellular ATP levels, and enhances respiratory inhibitor-induced mitochondrial defects such as loss of membrane potential (Deltapsim), morphological abnormalities, and reductions in activities of complex I+III and complex II+III, as well as cell death. Taken together, these results suggest that Drob-1 is essential for neuronal cell function, and that Drob-1 protects neurons from expanded polyglutamine-mediated neurodegeneration through the regulation of mitochondrial homeostasis (Senoo-Matsuda, 2005).
This report shows that Drob-1 can either promote (cell death during embryogenesis) or inhibit (polyglutamine-induced neurodegeneration) cell death, depending on a variety of conditions. In addition, it was shown that Buffy also has dual functions, namely a survival function (for cell death during embryogenesis) and a proapoptotic function (for polyglutamine-induced neurodegeneration) (Senoo-Matsuda, 2005).
Certain members of the Bcl-2 family proteins can function as both anti- and prodeath factors. Antiapoptotic Bcl-2 and Bcl-xL can be converted into proapoptotic proteins when they are cleaved by caspases or by other proteases. The resulting C-terminal fragments have a 'Bax-like' prodeath activity that induces cytochrome c release from mitochondria and forms pores in synthetic membranes. C. elegans CED-9 also exhibits prodeath as well as antideath activity. Proapoptotic Bax and Bak may promote or inhibit neuronal death depending on the specific death stimulus, neuron subtype, and stage of postnatal development. Bax promotes the survival of trigeminal ganglia neurons during development in mice that are deficient in NGF or TrkA, while it promotes the death of superior cervical ganglia neurons in the same models. Bax potently protects mice and cultured hippocampal neurons from Sindbis virus-induced apoptosis, whereas it promotes the death of Sindbis virus-infected dorsal root ganglia neurons. Bak protects hippocampal neurons from the cell death caused by excitotoxicity or viral infection; however, as mice mature, Bak function is converted from anti- to prodeath in virus-infected spinal cord neurons. Bak also protects mice from kainate-induced seizures, suggesting a possible role in regulating synaptic activity. Drob-1 has also been shown to have a protective activity against serum-deprivation- or CED-3-induced S2 cell death. Thus, individual Bcl-2 family proteins can have a pro- or antiapoptotic function, depending on the cellular context or specific stimulus. These findings, combined with the observations in this study, suggest that the dual-function nature of Bcl-2 family proteins may be evolutionarily conserved from nematodes to mammals (Senoo-Matsuda, 2005 and references therein).
Mitochondrial function [i.e., the production of ATP, regulation of apoptosis, and production of reactive oxygen species (ROS)] is crucial for the maintenance of postmitotic tissues (e.g., muscles and brain) in normal aging, and plays a role in degenerative diseases in humans and in animal models. During normal aging and the progression of human degenerative diseases, a decrease in the total number of cells in some postmitotic tissues (e.g., heart, skeletal muscle, and brain) is associated with a reduction in mitochondrial metabolic activity. This study shows that Drob-1 plays an important role in the survival of postmitotic neurons under both physiological and pathological conditions. Importantly, the finding that downregulation of Drob-1 results in a decrease in cellular ATP levels suggests that Drob-1 may be involved in the maintenance of mitochondrial metabolism. In addition, Drob-1 protects cells from stresses that cause mitochondrial dysfunction. Thus, these results suggest that Drob-1 may regulate the homeostasis of neurons and the aging process by maintaining mitochondrial metabolism. The ubiquitin-proteasome system plays a crucial role in preventing the polyglutamine-induced accumulation of unfolded proteins. This system acts in an ATP-dependent manner. Inhibition of the mitochondrial respiratory chain by the complex I inhibitor rotenone reduces the ubiquitin-proteasomal activity in both rat primary dopaminergic neurons and human SH-SY5Y neuroblastoma cells. Expanded polyglutamine protein has been reported to cause mitochondrial dysfunction, ATP loss, a defect in complex II enzyme activity, and subsequent inhibition of the ATP-dependent ubiquitin-proteasome system. These results lead to the proposal of a model in which Drob-1 suppresses polyglutamine-induced ATP depletion, thereby facilitating the subsequent activation of the ubiquitin-proteasome system, which protects neurons from cell death and degeneration. Buffy can antagonize this survival function of Drob-1 in neurons (Senoo-Matsuda, 2005).
Downregulation of Drob-1 induces ATP depletion and a shorter lifespan in flies. In C. elegans, mitochondrial complex II deficiency causes a shorter lifespan, hypersensitivity to oxidative stress, energy depletion, ROS overproduction, and CED-3- and CED-4-dependent supernumerary cell death (Ishii, 1998; Senoo-Matsuda, 2001; Senoo-Matsuda, 2003). In the complex II-deficient C. elegans mutant mev-1, the shorter lifespan is partially rescued by a loss-of-function mutation of CED-3, suggesting that the supernumerary apoptosis may contribute to shortening the lifespan in C. elegans (Senoo-Matsuda, 2003). Interestingly, the shorter lifespan in the mev-1 mutant may be associated with a decrease in the mitochondrial localization of CED-9 and its downregulation (Senoo-Matsuda, 2003). In mammals, Bcl-xL can prevent the perturbation of mitochondrial ATP/ADP exchange caused by growth factor deprivation, and can maintain oxidative phosphorylation under the growth-factor-withdrawal condition. The proapoptotic Bcl-2 family protein Bad is required to assemble the mitochondria-based glucokinase complex, which regulates glycolysis. Thus, the regulation of mitochondrial homeostasis may be an evolutionarily conserved role of Bcl-2 family proteins (Senoo-Matsuda, 2005).
The findings also suggest that Bcl-2 family proteins may play a crucial role in the pathogenesis of polyglutamine diseases. It would be greatly informative to determine whether Bcl-2 family proteins also play a crucial role in mammalian systems that can be a therapeutic target for neurodegenerative disorders. Further study of Drob-1 should increase understanding of the universal roles of Bcl-2 family proteins and may contribute to the development of new therapeutic applications, not only for polyglutamine diseases, but also for other abnormal-protein-accumulating neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (Senoo-Matsuda, 2005).
Bcl-2 family members are pivotal regulators of programmed cell death (PCD). In mammals, pro-apoptotic Bcl-2 family members initiate early apoptotic signals by causing the release of cytochrome c from the mitochondria, a step necessary for the initiation of the caspase cascade. Worms and flies do not show a requirement for cytochrome c during apoptosis, but both model systems express pro- and anti-apoptotic Bcl-2 family members. Drosophila encodes two Bcl-2 family members, Debcl (pro-apoptotic) and Buffy (anti-apoptotic). To understand the role of Debcl in Drosophila apoptosis, authentic null alleles at this locus were produced. Although gross development and lifespans were unaffected, it was found that Debcl was required for pruning cells in the developing central nervous system. debcl genetically interacted with the ced-4/Apaf1 counterpart dark, but was not required for killing by RHG (Reaper, Hid, Grim) proteins. debclKO mutants were unaffected for mitochondrial density or volume but, surprisingly, in a model of caspase-independent cell death, heterologous killing by murine Bax required debcl to exert its pro-apoptotic activity. Therefore, although debcl functions as a limited effector of PCD during normal Drosophila development, it can be effectively recruited for killing by mammalian members of the Bcl-2 gene family (Galindo, 2009).
Using a targeted recombination strategy, seven allelic strains were recovered that were definitively amorphic for debcl. These studies exclude a general requirement for debcl as a global apoptotic effector, which had been suggested from gene silencing analyses. Nevertheless, three compelling lines of evidence establish that debcl does function to regulate a limited number of developmental cell deaths. (1) In every allelic combination tested, TUNEL labeling was consistently and markedly reduced. (2) In every allele tested, debcl genetically interacted with a hypomorphic allele of the apoptosomal gene dark. (3) Extra cells in debcl embryos were detected using markers that visualize persisting or 'undead' cells in canonical PCD mutants. Although the impact caused by eliminating debcl was modest, it is noted that reproducible and consistent PCD phenotypes were observed for all alleles tested. Furthermore, it is also worth noting that the data reflect counts of marked cell populations, only a small fraction of which actually die. Hence, if only the cells that are lost are considered and these are compared against benchmarks seen in animals completely defective for PCD, the effects caused by eliminating debcl are substantial. For example, in H99 animals where no PCD occurs, a 37% excess of Kr+ cells is seen in Bolwig's organ and, by comparison, an excess of up to 23% Kr+ cells was seen in debcl mutants. Graded effects along the anteroposterior axis is another familiar phenotype seen here, and, likewise, has been reported for persisting motoneurons in H99 mutants. Specifically, in cases where extra neuronal cells were observed (e.g. α-Kruppel, these cells tended to appear more commonly among the posterior segments. This trend of extra cells in more posterior segments is consistent with previous studies reporting the prominence of neuronal degeneration in the abdominal ganglion. Segments with supernumerary cells averaged 17% additional cells in debclKO animals and, by comparison, mutations in the apoptosomal genes dark and dronc produced a range from 33%-50% excess cells. From these combined results, it is speculated that perhaps debcl augments apoptotic signaling in certain cell types. Not all tissues were impacted in debclKO animals, however. Notable examples of Drosophila PCD that were unaffected by debcl status included PCD of interommatidial cells in the eye, PCD of the salivary gland and elimination of Crz+ neurons during larval to pupal transition, as well as of markers specific for motoneurons during embryogenesis. Hence, to the extent represented by the markers that were studied, the impact of debcl status on Drosophila cell death appears to be limited to certain embryonic neurons and glia, but not on the PCD of motoneurons. Taken together, these studies exclude a universal requirement for debcl in PCD, yet establish a limited role for this gene in the death of certain cell (Galindo, 2009).
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