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Previous Article | Next Article 
Molecular and Cellular Biology, November 2004, p. 9848-9862, Vol. 24, No. 22
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.22.9848-9862.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Signal Transduction Laboratory, Cancer Research UK London Research Institute,1 Neurometabolic Unit,6 Division of Neuropathology and Department of Neurodegenerative Disease,8 Institute of Neurology, London, Cell Death Regulation Laboratory, MRC Toxicology Unit, Leicester,2 Department of Comparative Genomics, GlaxoSmithKline, Harlow, Essex, United Kingdom,3 Department of Comparative Genomics, GlaxoSmithKline, Upper Providence, Philadelphia, Pennsylvania,5 Department of Neurodegeneration and Neurorestoration, Center of Neurology and Center of Molecular Physiology of the Brain, University of Göttingen, Göttingen, Germany,4 Division of Cellular and Molecular Biology, AMDI Institute, Toronto, Ontario, Canada7
Received 4 August 2004/ Accepted 10 August 2004
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ABSTRACT |
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INTRODUCTION |
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Smac/DIABLO is another mammalian mitochondrial protein containing an amino-terminal Reaper motif that has been identified due to its ability to interact with and antagonize IAPs (2, 34). When overexpressed, Smac/DIABLO can sensitize cells to apoptotic stimuli. However, there is relatively little evidence that endogenous Smac/DIABLO plays an important physiological role in the regulation of apoptosis (33) and mice with the gene for this protein deleted show no detectable abnormalities (19). Cells derived from these Smac/DIABLO knockout mice also display normal apoptosis regulation. Similarly, mice with a deletion of the gene for the broadly expressed IAP family member XIAP show no abnormal phenotype (6). It is possible that IAPs and their antagonists play relatively minor roles in the regulation of apoptosis in mammals, unlike in flies. Alternatively, the high degree of redundancy among IAPs, and possibly also among Reaper motif-containing proteins, may make their in vivo function hard to analyze by single-gene deletion experiments.
While Smac/DIABLO has no obvious catalytic function and no clear homologues (outside of the 4-amino-acid Reaper motif) in invertebrates, HtrA2/Omi is a member of a well-conserved family of PDZ domain-containing serine proteases that are found in most eukaryotes and prokaryotes. Well-characterized members of the family include DegS and DegP in E. coli. DegP is a protein that protects against heat stress and that acts as a chaperone for unfolded proteins at low temperatures, while acting to degrade them at elevated temperatures (25). DegS detects unfolded outer membrane porins in the periplasm, triggering a signaling protease cascade leading to adaptive changes in gene expression (36). The known protective stress response activities of bacterial HtrA2/Omi homologues thus appear to be very different from the proposed proapoptotic, Reaper-like action of mammalian HtrA2/Omi. However, it has been reported recently that the neurodegenerative phenotype leading to juvenile mortality in the Mnd2 strain of mice is caused by a protease-inactivating point mutation in the gene encoding HtrA2/Omi (12). These mice show loss of a population of striatal neurons (11), suggesting possible failure of a protective mechanism and providing circumstantial evidence that the protease activity of HtrA2/Omi might have a function in protein quality control akin to that of its bacterial homologues. On the other hand, the Mnd2 mice still express HtrA2/Omi protein with a correctly processed amino-terminal Reaper motif, so they are not expected to show the effect of removing the IAP-antagonizing activity of HtrA2/Omi.
In this paper we report the phenotype of mice with a homozygous deletion of the gene encoding HtrA2/Omi. Unlike Mnd2 mice, these animals lack all expression of HtrA2/Omi and hence have neither its Reaper motif nor its protease activity. The phenotype of HtrA2/Omi knockout mice is extremely similar to that of Mnd2 mice, with early death resulting from the loss of a population of striatal neurons, suggesting that while the serine protease activity of HtrA2/Omi has a protective function, the amino-terminal Reaper motif does not have an obvious nonredundant proapoptotic role. To address whether the removal of both the major XIAP binding and the Reaper motif-containing proteins would reveal a physiological proapoptotic function for mammalian Reaper-related proteins, HtrA2/Omi knockout mice were crossed with animals in which the gene encoding Smac/DIABLO had been deleted. Loss of Smac/DIABLO did not further exacerbate the phenotype of the HtrA2/Omi knockout mice. We conclude that if Reaper motif-containing proteins play any physiological role in apoptosis regulation in mice, it is very highly redundant and must be mediated in addition by proteins other than the major reported IAP interactors. HtrA2/Omi, on the other hand, has a significant protective function that requires its protease activity and may be related to that of the homologous bacterial stress-adaptive proteins DegP and DegS.
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MATERIALS AND METHODS |
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Cell culture. Primary mouse embryo fibroblasts (MEFs) were established from E14.5 embryos according to standard procedures (10). The cells were maintained in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum, L-glutamine, and antibiotics. To establish immortalized MEFs overexpressing the simian virus 40 large T antigen, primary MEFs were cultured in the presence of viral supernatants prepared by transfecting the Phoenix ecotropic retrovirus packaging cell line with pBabe Puro SV40LT. Cells that integrated the virus were selected in medium containing 2 µg of puromycin per ml (Sigma). Thymocytes were prepared from HtrA2/Omi+/+ and HtrA2/Omi–/– mice by standard procedures (22) and plated at 5 x 106/ml in 24-well plates in RPMI medium containing 10% fetal calf serum. Primary cortical neurons were isolated from E14.5 embryos and seeded at a density of 5 x 105 per well in 12-well plates coated with poly-L-lysine. Cells were maintained in Neurobasal medium (Invitrogen) supplemented with B27, Glutamax, and penicillin-streptomycin (Invitrogen). Following 3 days in culture, contamination from fibroblasts and glial cells was reduced by supplementing culture media with 10 µM cytosine arabinoside (Sigma). Cells were cultured for 10 days, with exchange of 50% of the medium volume with fresh medium every 2 days before treatment with toxic stimuli.
Inclined-platform test. Mice were placed in the middle portion of a 60o-inclined platform, and evaluation was performed as previously described (23).
Histology and immunohistochemistry. Brain tissues were harvested from 20- and 30-day-old mice and fixed in 10% formalin, embedded in paraffin, cut into 3-µm-thick sections, and processed for hematoxylin-eosin staining. In addition, immunostaining with the following stains was carried out according to the manufacturer's instructions: glial fibrillary acidic protein (GFAP; rabbit polyclonal antibody; dilution, 1:300; DAKO), NeuN (antineuronal nuclei; mouse monoclonal antibody; dilution, 1:4,000; Chemicon), MAP2 (microtubule-associated protein-2 mouse monoclonal antibody; dilution, 1:500; Chemicon), calbindin (rabbit polyclonal antiserum; dilution, 1:200; Chemicon), and tyrosine hydroxylase (TH; rabbit polyclonal antiserum; dilution, 1:1,000; Chemicon).
Biotinylated secondary antibodies were used for all staining, and visualization was with a horseradish peroxidase-conjugated streptavidin complex and diaminobenzidine as a chromogen. All immunostaining was carried out with the automated Nexus staining apparatus (Ventana Medical Systems) according to the manufacturer's guidelines. Photographs were obtained on a ColorView II digital camera (Soft Imaging System) mounted on a Zeiss Axioplan microscope and composed in Adobe Photoshop.
Stereological cell counts. Immunohistochemistry was performed on serial sections (50 µm thick) of the striatum after the tissue had been fixed with paraformaldehyde cryoprotected in 30% (wt/vol) sucrose in phosphate buffer and frozen rapidly in liquid isopentane. Counts were performed by using the optical fractionator method as described previously (39). In agreement with this method, NeuN-positive neurons were counted in the left striatum of every third section throughout the entire extent of the striatum. Each section was viewed at low power (x2.5 objective), and the striatum was outlined. Then the number of NeuN-positive cells in the various groups of animals were counted at high power (x63 oil immersion lens). In another set, the first and fourth sections from the striatum, just lateral of the thalamus, in a relatively posterio-medial portion of the basal ganglia were outlined and cells in the lower half were counted with a stereological program (StereoInvestigator; Microbrightfield, Williston, Vt.). TH immunostaining was carried out on striatal and midbrain sections, and the TH-stained substantia nigra pars compacta neurons were counted by stereology using the optical fractionator method. The striatal density of TH immunoreactivity was determined as described previously (28).
Determination of mitochondrial enzyme activities. A 4-mm-thick coronal slice of each mouse brain containing the medial striatum was isolated with a razor blade. The striatum was microdissected, placed in dry ice, and stored at –80°C until analysis. Prior to analysis, samples were homogenized at a concentration of 10% (wt/vol) in 10 mM Trizma base-1 mM EDTA and subjected to three cycles of freezing and thawing to lyse membranes. All activities were determined at 30°C. Enzyme activities were assessed using a Uvikon 940 spectrophotometer (Kontron Instruments Ltd., Watford, United Kingdom). Complex I (NADH ubiquinone reductase) activity was measured according to the method of Ragan and colleagues (20). Complexes II and III (succinate cytochrome c reductase) were measured according to the method of King (14). Complex IV (cytochrome c oxidase) was measured according to the method of Wharton and Tzagoloff (37). Citrate synthase was measured as described by Shepherd and Garland (24).
Morphological analysis of mitochondria. Wild-type and HtrA2/Omi knockout MEFs were cultivated in Dulbecco modified Eagle medium containing 10% fetal bovine serum until confluence. Cells were then subjected to the following treatments for 30 min: 25 µM m-chlorocarbonyl cyanide phenylhydrazone (CCCP; Calbiochem) or 25 µM rotenone (Calbiochem). Following treatment, the medium was replaced and cells were further incubated at 37°C for 2 h. Following incubation, cells were processed for electron microscopy by gently replacing the culture medium with fixative solution (4% paraformaldehyde, 5% glutaraldehyde, and 5 mM CaCl2 in 0.2 M cacodylate buffer at pH 7.4) and incubating the mixture for 1 h at room temperature. Cells were postfixed with 1% osmium tetroxide-1% potassium ferrocyanide for 1 h at room temperature. Fixed cells were stained with 5% aqueous uranyl acetate overnight at room temperature and harvested by scraping. The resulting pellet was dehydrated and embedded in TAAB embedding resin. Ultrathin sections were stained with lead citrate and examined in a Jeol 100-CXII electron microscope equipped with a rotating-stage and eucentric goniometer. All quantitative assessments of mitochondrial morphology were based on scoring a minimum of 25 cells for each treatment. For each treatment, the number of mitochondria showing specific changes was determined.
Induction of cell death in MEFs, thymocytes, and primary neurons.
For induction of cell death in MEFs, cells (5 x 104 per well) were plated in 12-well plates and treated 24 h later with UV, etoposide (Sigma), or tumor necrosis factor alpha (TNF-
) (Roche) in the presence of 5 µg of cycloheximide (Sigma) per ml. Cells were harvested after 16 h. Cell viability was determined by staining cells with propidium iodide (Molecular Probes) according to the manufacturer's instructions, followed by flow cytometric analysis. For thymocyte cell death analysis, cells were incubated for 16 h in the presence or absence of anti-Fas antibody (Jo2; BD-Pharmingen), etoposide (Sigma), CCCP (Calbiochem), or rotenone (Calbiochem). Cells were harvested after 16 h, and viability was determined as described above. Immortalized MEFs were plated (2 x 105 per well) in six-well plates and treated 16 h later with CCCP, rotenone, tunicamycin (Sigma), or hydrogen peroxide (Sigma) for 27 h. DNA content was determined by staining cells with propidium iodide, followed by flow cytometric analysis as previously described (1). For neuron cell death analysis, cells were incubated in the presence or absence of glutamate (Sigma) for 4 h, fixed in 4% paraformaldehyde, and stained with Hoechst 33342 dye (Molecular Probes) according to the manufacturer's instructions, and nuclear chromatin was scored as being condensed or not condensed by fluorescence microscopy.
Immunoprecipitation of Smac/DIABLO complexes. MEFs derived from HtrA2/Omi+/+ and HtrA2/Omi–/– animals were lysed in phosphate-buffered saline containing 1% Triton X-100 and a 1-µg/ml concentration (each) of chymostatin, leupeptin, antipain, and pepstatin A. Cell lysates were cleared by centrifugation at 10,000 x g and incubated in the presence of anti-Smac antibody (567360, 50 ng/ml; Calbiochem) in phosphate-buffered saline-1% Triton X-100. Antigen-antibody complexes were allowed to form at 4°C for 1 h and then precipitated using protein G-Sepharose beads. Proteins were resolved in a sodium dodecyl sulfate-12% polyacrylamide gel and transferred to an Immobilon-P membrane (Millipore). Immunoblot analysis was performed with the following antibodies: anti-Smac (567360; Calbiochem), anti-HtrA2/Omi as previously described (17), and anti-XIAP (AAM-050; Stressgen).
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RESULTS |
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Deletion of HtrA2/Omi results in a mitochondrial dysfunction. The serine protease HtrA2/Omi is localized to the intermembrane space of mitochondria of healthy cells. In order to determine if HtrA2/Omi's absence resulted in a compromise of mitochondrial metabolism, we analyzed at the biochemical level the components of the mitochondrial electron transport chain in extracts prepared from the striata of HtrA2/Omi+/+ and HtrA2/Omi–/– animals. This analysis failed to reveal any significant compromise of the function of complex I, II, III, or IV (Fig. 5A) when enzymatic activities were normalized against the activity of the mitochondrial matrix enzyme citrate synthase, to account for differences in mitochondrial density in the samples. However, extracts prepared from HtrA2/Omi knockout animals showed a significant decrease of the yield of mitochondrial citrate synthase (Fig. 5A), indicating that mitochondrial density is likely to be reduced in tissues derived from knockout animals.
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Deletion of HtrA2/Omi results in increased susceptibility to cell death stimuli. The observation that mitochondria in cells lacking HtrA2/Omi displayed an increased susceptibility to cellular stresses led us to ask whether this increased sensitivity could affect cell viability. We therefore subjected thymocytes from control and HtrA2/Omi knockout animals to classical apoptosis-inducing agents that activate both the intrinsic and extrinsic pathways, as well as drugs that specifically target mitochondria. Our results indicate that although cells lacking HtrA2/Omi do not show an altered sensitivity to agents that trigger the extrinsic cell death pathway, such as Fas agonistic antibodies (Fig. 6A), they display increased sensitivity to agents that trigger apoptosis through the intrinsic pathway, such as the DNA-damaging agent etoposide (Fig. 6B), and agents that perturb mitochondrial function, such as CCCP and rotenone (Fig. 6C and D). Analysis of immortalized MEFs derived from either wild-type or HtrA2/Omi knockout animals was consistent with an increased sensitivity of cells lacking HtrA2/Omi to mitochondrial stress stimuli as well as agents that cause both oxidative stress (hydrogen peroxide) and endoplasmic reticulum stress (tunicamycin) (Fig. 6E).
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Endogenous Smac/DIABLO is capable of sequestering all XIAP in cells lacking HtrA2/Omi. The results presented so far indicate that, rather than protecting cells from apoptosis, the lack of HtrA2/Omi results in increased sensitivity to some death stimuli. It is possible that the presence of the other major Reaper motif-containing protein, Smac/DIABLO, might compensate for the removal of HtrA2/Omi in terms of its ability to neutralize IAPs. To explore this possibility, we immunoprecipitated Smac/DIABLO from lysates of MEFs from wild-type or HtrA2/Omi knockout mice with close to 100% efficiency (Fig. 7). Removal of Smac/DIABLO resulted in the depletion of the major portion of the caspase inhibitor XIAP from wild-type cell lysates and almost all of XIAP from HtrA2/Omi knockout cell lysates (Fig. 7, upper panel, middle section, which shows the amount of XIAP in cell lysates from which Smac/DIABLO has been immunodepleted). This depletion indicates that there is sufficient Smac/DIABLO present in cells to compensate for the loss of HtrA2/Omi in terms of its potential ability once released from mitochondria to bind XIAP and neutralize its caspase-inhibitory activity.
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, revealed that the levels of cell death in wild-type and double-knockout cells were identical (Fig. 8C). However, as was observed in cells lacking solely HtrA2/Omi, cells lacking both HtrA2/Omi and Smac/DIABLO displayed increased sensitivity to apoptosis induction by the DNA-damaging agent etoposide (Fig. 8D). We conclude that deletion of both genes for the mammalian IAP interactors HtrA2/Omi and Smac/DIABLO does not result in resistance to apoptosis in the cell types analyzed but rather in an increased sensitivity to some death-inducing agents. |
DISCUSSION |
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Why might the loss of these two Reaper motifs have so little effect in vivo? The possibility that the Reaper/IAP system is not a major regulator of cell death in mammals cannot be ruled out. While clearly of great importance in flies, the system does not exist in worms. Although at least seven mammalian IAPs have been identified, it could be argued that no true Reaper, Hid, or Grim analogues have been found. On the other hand, it is also possible that an effect might become apparent later in life in mice with a healthy genetic background whose life spans are not restricted to approximately 4 weeks due to the loss of HtrA2/Omi protease activity. In order to address this possibility rigorously, it will be necessary to create mice in which the Reaper motif (AVP) of HtrA2/Omi has been replaced by a gene with a sequence unable to bind to IAPs knocked in, such as GVP. A third possibility is that, even though Smac/DIABLO and HtrA2/Omi are the major IAP binding proteins in most cells studied, there may exist a much greater degree of redundancy in Reaper motif-containing proteins than previously suspected. It has been suggested that caspase cleavage of proteins may frequently generate novel amino termini with alanine residues that may be able to bind to IAPs in a Reaper-like manner (9). This possible ability to bind IAPs might provide feed forward amplification of caspase activation once activation is under way, although it seems unlikely to be able to promote the initiation of caspase activation. It is also possible that many mitochondrial proteins might have some form of amino-terminal Reaper motif (32). In this model, severe mitochondrial damage would cause the release of many proteins with the ability to antagonize IAPs, not just Smac/DIABLO and HtrA2/Omi, so the loss of the Reaper motifs of these two may have little impact. The reason for the possible frequent appearance of amino-terminal Reaper motifs in mitochondrial proteins may relate to the N end rule (31), under which amino-terminal alanine residues are destabilizing. Thus, a low-level release of mitochondrial proteins into the cytosol during normal cell function may be mopped up effectively by the proteasome machinery.
Data presented here in Fig. 7 show that Smac/DIABLO is clearly the most efficient XIAP binding protein in detergent lysates of cells: in the absence of HtrA2/Omi, all XIAP is complexed to Smac/DIABLO. In the presence of HtrA2/Omi, a minor fraction of XIAP is not complexed to Smac/DIABLO, presumably because it is bound to HtrA2/Omi. Thus, if there are other physiological Reaper motif-containing XIAP binding proteins in cells, it is unlikely that they will be found readily in the presence of competing Smac/DIABLO. However, it should be noted that all the XIAP complexes seen in coimmunoprecipitations from detergent cell lysates must have been formed after cell and organelle disruption. Another Reaper motif-containing XIAP binding protein has been identified, GSPT1/eRF3 (7), although this protein is not localized to the mitochondria and so would be unlikely to contribute to caspase regulation in the manner proposed for HtrA2/Omi and Smac/DIABLO.
If the Reaper motif at the processed amino terminus of HtrA2/Omi does not play a major nonredundant role in cell death regulation, what can be concluded about the function of the protease domain of HtrA2/Omi? As in Mnd2 mice, a population of neurons in the striata of HtrA2/Omi knockout mice can be seen to be lost from about 20 days of age. In the case of the Mnd2 mice, this cell death, and the mobility defect, is not blocked by Bcl2 overexpression, suggesting that apoptosis is not the sole cell death mechanism at work (21). Astrogliosis is detected by GFAP staining in this area. Neuronal cell populations isolated from the knockout mice are more sensitive than wild-type controls to excitotoxicity induced by glutamate. Nonneuronal cell types, such as fibroblasts and T lymphocytes, from the HtrA2/Omi knockout mice also show increased sensitivity to induction of cell death by DNA-damaging agents and inhibitors that target the mitochondria, such as the complex I inhibitor rotenone and the uncoupler CCCP.
Since HtrA2/Omi knockout mice develop a phenotype with parkinsonian features starting at about day 25 (unpublished data), it will be interesting to analyze patients with Parkinson's disease and parkinsonian syndromes for HtrA2/Omi deficits. We did not detect a degeneration of TH-positive dopaminergic neurons in the substantia nigra pars compacta, as occurs in Parkinson's disease patients. However, our data show a presynaptic and postsynaptic defect of the nigrostriatal pathway in the striatum: in a frontal area of the striatum there is a loss of neurons as well as a decrease of TH-positive fibers. This finding suggests similarity to some parkinsonian syndromes.
While HtrA2/Omi is ubiquitously expressed, it seems that a localized population of striatal neurons are uniquely dependent on its protease function. It is possible that HtrA2/Omi acts to protect these cells against some forms of pathogenic stress. Stresses that have been implicated in the death of neurons in vivo include oxidative stress, excitotoxic stress, and the accumulation of denatured proteins, including those with expanded polyglutamine repeats. Due to its mitochondrial localization, HtrA2/Omi probably acts to protect these organelles, and in particular the mitochondrial intermembrane compartment, from damage resulting from the stresses to which striatal neurons are normally exposed. In this regard, the similarity to the bacterial HtrA homologues, especially DegS, is particularly striking. This protein has exactly the same domain structure as HtrA2. It is localized in the periplasmic space between the inner and outer bacterial cell membranes, with its amino-terminal region inserted into the inner membrane: this localization is clearly analogous to that of full-length HtrA2/Omi within the mitochondria. The appearance of the exposed C termini of unfolded porins, outer membrane channel proteins, in the periplasmic space results in activation of the protease domain of DegS due their binding to its PDZ domain (36). Similarly, engagement of the PDZ domain of HtrA2/Omi results in activation of its protease activity (15, 18), although in the case of HtrA2/Omi its natural PDZ binding partners within the mitochondria remain to be identified. Once activated, DegS cleaves RseA, an inner membrane protein which, together with RseB, sequesters the transcriptional regulator 
E at the cytoplasmic face of the inner membrane. It is interesting that DegS cleaves RseA between a valine and a serine (36), similar to the optimal peptide substrate cleavage for human HtrA2/Omi (18). Cleavage of RseA by DegS leads to further proteolysis by the intramembrane protease YaeL, resulting in the release of E into the cytosol to induce the expression of periplasmic stress response genes (36). While the similarity between HtrA2/Omi and DegS is evident, it is not possible to identify obvious mammalian homologues of other downstream components of the bacterial periplasmic stress response system.
If HtrA2/Omi does act in a manner similar to that of DegS, it is at present unclear exactly what cellular stresses might stimulate the protease activity of HtrA2/Omi. The exact nature of the cell damage caused in striatal neurons lacking HtrA2/Omi is also unknown: there is no obvious defect in total brain tissue in the functions of the different mitochondrial electron transport complexes or the generation of reduced glutathione. However, it is possible that these could be altered in a small population of neurons that would be difficult to detect by biochemical assay of whole tissue extracts. The main effect on mitochondrial function seen in liver and in cultured fibroblasts from the HtrA2/Omi knockout mice is the reduction in mitochondrial membrane potential relative to that of controls, as was also reported for Mnd2 mice (12). If it acts like DegS, the loss of HtrA2/Omi may result in an inability to mount an adaptive transcriptional response under stress conditions that cause protein unfolding, leaving the mitochondria of striatal neurons unable to maintain their membrane potential and rendering the cells prone to either necrotic or apoptotic death. Alternatively, if HtrA2/Omi functions like another E. coli homologue, DegP (25), mitochondria from knockout mice may be unable to degrade unfolded proteins in the intermembrane space under conditions of cell stress, and these could form aggregates that would directly compromise the maintenance of the mitochondrial membrane potential. Which of these different models of HtrA2/Omi's neuroprotective function is correct may be clarified once its mitochondrial binding partners are identified and once differences in stress-induced transcriptional responses are determined.
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ACKNOWLEDGMENTS |
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Kristina Klupsch was funded by a studentship from the Boehringer Ingelheim Fonds.
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FOOTNOTES |
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