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Clk-1 deficiency induces apoptosis associated with mitochondrial dysfunction in mouse embryos
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Mechanisms of Ageing and Development 129 (2008) 291–298 www.elsevier.com/locate/mechagedev
Clk-1 deficiency induces apoptosis associated with mitochondrial dysfunction in mouse embryos Mayumi Takahashi a, Takahiko Shimizu a, Eiko Moriizumi a, Takuji Shirasawa a,b,* a
Research Team for Molecular Biomarkers, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173-0015, Japan b Department of Aging Control Medicine, Juntendo University Graduate School of Medicine, Bunnkyou-ku, Tokyo 113-0033, Japan Received 14 August 2007; received in revised form 11 December 2007; accepted 27 January 2008 Available online 12 February 2008
Abstract Clk-1 gene encodes demethoxyubiquinone hydroxylase that catalyzes the production of coenzyme Q (CoQ) in mitochondria. Clk-1-deficient mice that lack CoQ fail to survive beyond the embryonic day 10.5 (E10.5). However, the relationship between the clk-1-deficiency and embryonic lethality remains unclear. We show in this study that TUNEL-positive cells are frequently observed in whole bodies of clk-1-deficient mouse embryos at E10.5. In addition, dissociated cells from the embryos exhibited characteristic features of apoptosis, such as externalization of phosphatidylserine on the plasma membrane, caspase-3 activation, and the release of cytochrome c from mitochondria into the cytoplasm, as the first sign of mitochondria-mediated apoptosis. In embryonic cells, the mitochondrial functions such as maintenance of the mitochondrial membrane potential and intracellular ATP level were impaired. Since exogenous CoQ10 rescued the mitochondrial dysfunction and suppressed apoptosis in clk-1-deficent cells, we propose that clk-1-deficency induces apoptosis associated with mitochondrial dysfunction due to a lack of CoQ, which may lead to embryonic lethality in mice around E10.5. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Clk-1; Apoptosis; Embryonic lethality; Coenzyme Q; Mitochondria; Mouse embryo
1. Introduction Clk-1 was initially reported in Caenorhabditis elegans as a gerontogene that is evolutionarily conserved among eukaryotes from yeast to human (Ewbank et al., 1997; Asaumi et al., 1999; Vajo et al., 1999; Takahashi et al., 2001; Jonassen et al., 1996) and in some bacteria as well (Stenmark et al., 2001; Andersson et al., 1998) and encodes demethoxyubiquinone (DMQ) hydroxylase that converts DMQ to 5-hydroxy-ubiquinone in the biosynthesis of coenzyme Q (CoQ)/ubiquinone (Marbois and Clarke, 1996). All the clk-1 mutants of yeasts, nematodes, and mice fail to synthesize CoQ, but instead accumulate DMQ (Jonassen et al., 2001; Levavasseur et al., 2001; Nakai et al., 2001). Among the functions of CoQ, the most pivotal one is to
* Corresponding author at: Department of Aging Control Medicine, Juntendo University Graduate School of Medicine, 3-3-10-201 Hongou, Bunnkyou-ku, Tokyo 113-0033, Japan. Tel.: +81 3 3814 1134; fax: +81 3 3814 1134. E-mail address: [email protected] (T. Shirasawa). 0047-6374/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2008.01.008
shuttle electrons from complex I or II to III in the mitochondrial respiratory chain, thereby leading to maintenance of mitochondrial membrane potential (DCm) and the production of ATP by oxidative phosphorylation (Ernster and Dallner, 1995; Turunen et al., 2004). Clk-1 mutants of C. elegans exhibit a long lifespan (Wong et al., 1995; Lakowski and Hekimi, 1996) when supplied with CoQ in their diet (Jonassen et al., 2001), but their development is arrested at the L2 larval stage when fed with mutant E. coli, deficient in CoQ (Jonassen et al., 2001). To investigate whether clk-1 plays a role in the embryonic development or determination of lifespan in mammals, we generated clk-1deficient mice (Nakai et al., 2001). Clk-1-deficient mice fail to survive beyond embryonic day 10.5 (E10.5) (Nakai et al., 2001; Levavasseur et al., 2001). However, the mechanism by which clk-1-deficiency leads to embryonic lethality remains to be elucidated. In this study, we examined this problem using clk-1deficient mouse embryos and cells isolated from the embryos just before death. A significant reduction in mitochondrial respiratory function and frequent mitochondria-mediated
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apoptosis were induced in the clk-1-deficient mouse embryos or cells. Since exogenous CoQ10 rescued the mitochondrial dysfunction and suppressed the induction of apoptosis, we suggest that clk-1 plays a crucial role in the maintenance of mitochondrial respiratory function and cell survival through the production of CoQ during the early development of mice. 2. Materials and methods 2.1. Animals Mice used in this study were F1 hybrids of the C57BL/6CrSlc strain (Nakai et al., 2001) and ICR strain (Japan SLC, Inc.). Animals were housed in specific pathogen-free facilities on a 12-h light/dark cycle and were fed ad libitum. All protocols for animal use and experiments were reviewed and approved by the Animal Care Committee of the Tokyo Metropolitan Institute of Gerontology.
2.2. Cell preparation and treatment Embryos at E10.5 were dissected from the yolk sac and amnion, and digested with 2.4 U/ml Dispase I (Roche, Switzerland) and 10 mg/ml DNase I (Sigma) for 30 min at 37 8C in a CO2 incubator. The digestion was stopped by adding serum-free COSMEDIUM 001 (COSMO BIO Co., Ltd.) containing 100 U/ml penicillin and 100 mg/ml streptomycin. Dissociated cells were collected by centrifugation at 700 g for 5 min (Beckmann, CS-6KR) and resuspended in COSMEDIUM. In some experiments, dissociated cells were treated with 25 mM water-soluble CoQ10 (sCoQ10, Aqua Q10L10, Nisshin Pharma Inc., Japan) or placebo (Nisshin Pharma Inc., Japan) at the same concentration of sCoQ10 dissolved in serum-free COSMEDIUM 001 for 5 h before each assay. Genotypes of mice or embryos were determined by PCR as described previously (Nakai et al., 2001).
2.3. Histological detection of apoptosis Mouse embryos at E8.5 and 10.5 were fixed in 20% (v/v) formalin, dehydrated, embedded in paraffin, and sectioned at 3 mm. TdT-mediated XdUTP nick end labeling (TUNEL) for the detection of apoptosis in embryos was performed using a TUNEL Label kit (Roche Molecular Biochemicals). Briefly, deparaffinized and rehydrated sections were incubated with proteinase K (20 mg/ml, Roche Molecular Biochemicals) for 60 min at 37 8C, rinsed twice with phosphate-buffered saline (PBS), and incubated in 0.3% H2O2-containing methanol for 10 min at room temperature to block the endogenous peroxidase (POD) activity. After being washed in PBS, sections were incubated with TUNEL reaction mixture containing terminal deoxynucleotidyl transferase in moisture overnight at 4 8C, washed again with PBS, and incubated with converter-POD solution for 1 h at room temperature. For the visualization of apoptotic signals, sections were incubated with 30 -diamino-benzidine (DAB) as a chromogen and counterstained with haematoxylin. The apoptosis in mouse embryo was quantified using an annexin V-FITC Apoptosis Detection kit (Sigma). Dissociated cells from embryos at E10.5 or cells treated with sCoQ10 were stained with 0.5 mg/ml annexin V-FITC and 5 ng/ml Hoechst33342 (Calbiochem) for nuclear counterstaining in annexinbinding buffer, followed by fixation in 20% (v/v) neutralized formalin containing 0.25 mM CaCl2 at room temperature for 10 min in the dark. Fluorescence images were recorded through bandpass filters of 420–480 and 505–530 nm, and the numbers of both annexin-positive and total cells were counted for five distinct fields in each of five wells, respectively using a IN Cell Analyzer 1000 (GE Healthcare Bio-Sciences Corp.). The same experiment was repeated three times. Annexin-positive cells are expressed as a percentage of the total number of nuclei.
2.4. Immunocytochemistry Cells dissociated from mouse embryo at E10.5 were cultured on glassbottom dishes (Matsunami Glass Ind. Ltd.) with serum-free COSMEDIUM in a
humidified atmosphere of 5% CO2 in air at 37 8C for 4 h and fixed in a solution of 50% (v/v) methanol/50% (v/v) acetone (20 8C) for 5 min on ice. After being incubated in blocking buffer (10% goat serum in PBS) for 30 min at room temperature, cells were incubated with anti-cytochrome c antibody (BD PharMingen) overnight at 4 8C, followed by Alexa Fluor 546-conjugated goat anti-mouse secondary antibody (Molecular Probes, Inc., Eugene, OR) for 1 h at room temperature. Nuclei were counterstained with 5 ng/ml Hoechst33342. Finally, the samples were embedded in non-fluorescence glycerol (Merck) diluted with PBS and observed under a confocal laser scanning microscope (Zeiss LSM5 PASCAL), equipped with a blue diode and a He/Ne laser with excitation at 405 and at 543 nm, respectively. Fluorescence images were recorded through a band pass filter at 420–480 nm and a long pass filter at 580 nm, respectively.
2.5. Western blot analysis Mouse embryos at E10.5 were homogenized with sonication in ice-cold lysis buffer (10 mM Tris–HCl, containing 150 mM NaCl, 1% SDS and protease inhibitor cocktail, CompleteTM (Roche)). Homogenates were centrifuged at 10,000 g for 10 min at 4 8C, and the concentration of protein in the supernatant was measured with a DC Protein Assay Kit (Bio-Rad, Hercules, CA). Proteins (10–50 mg/treatment) were resolved by 15% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Immobilon-PSQ, Bio-Rad). The membrane was blocked with 5% milk in TBS/Tween (Tris-buffered saline with 0.05% (v/v) Tween 20) for 1 h, and incubated with anti-activated caspase-3 (Cell Signaling Tech., Inc., Danver, MA) (1:1000 in 5% milk TBS/Tween) overnight at 4 8C. The blot was washed with TBS/Tween and incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000, Gibco BRL) for 1 h at room temperature. After washing, the immunoreactive protein was detected with an ECL system (GE Healthcare Bio-Sciences Corp.) and Luminoimage Analyzer LAS-3000 (Fuji, Tokyo, Japan).
2.6. Determination of mitochondrial membrane potential Mitochondrial membrane potential (DCm) was measured using 5,50 ,6,60 tetrachloro-1,10 ,3,30 -tetraethylbenzimidazole carbocyanideiodide (JC-1, Calbiochem), a cationic cyanine fluorescence dye incorporated into mitochondria in proportion to DCm (Reers et al., 1991, 1995; Smiley et al., 1991). A stock solution of JC-1 was prepared at 2 mg/ml in dimethyl sulfoxide. Dissociated embryonic cells at E10.5 or cells treated with sCoQ10 were stained with a mixture of 1 mg/ml JC-1 and 5 ng/ml Hoechst33342 in culture medium for 10 min in a CO2 incubator, washed three times with Hanks’ solution, and observed under a confocal laser scanning microscope (Carl Zeiss). Fluorescence images of monomers and J-aggregates were simultaneously captured through a 505–535 nm bandpass filter and a 580 nm long pass filter, respectively. The nuclear staining with Hoechst33342 was detected with a 420–480 nm bandpass filter after excitation with a blue diode laser at 405 nm. The ratio of absorbance at 580–530 nm (580/530 ratio) was calculated as the relative DCm value.
2.7. Measurement of ATP levels The ATP levels in cells were determined using a bioluminescence assay kit (Promega Corp., Madison, WI). Dissociated embryonic cells were immediately collected by centrifugation and suspended in Hanks’ solution. In some experiments to examine the effect of CoQ on ATP production, dissociated cells were cultured in a 96-well-plate containing COSMEDIUM with or without 25 mM sCoQ10 for 5 h in a CO2 incubator. After cultivation, the plate was centrifuged at 2000 rpm for 5 min (KUBOTA 7800, RS-96S, Japan) and medium was discarded until 10 ml was left in each well. Cell suspensions just after dissociation or cultured cells in wells were incubated with the same volume of ATP assay reagent to induce cell lysis on a shaker for 10 min at room temperature in the dark. After all samples were measured with a luminometer (Lumat LB9507, Berthold Technologies, Bad Wildbad, Germany), background luminescence without cells was subtracted from each value. ATP standard curves were run in all experiments with different concentrations of ATP, and calculations were made from the curve. DNA content was assessed according to the method of Hinegardner (1971) with small modifications in parallel to
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normalize the data. Briefly, cells were fixed with 100% ethanol for 5 min, airdried, and incubated with 0.4% 3,5-diaminobenzoic acid dihydrochloride (Sigma) for 45 min at 60 8C. The reaction was stopped with 2N HCl and fluorescence was measured in each sample or standard (Ex. 400 nm, Em. 530 nm) with a fluorometer (SPECTRA MAX GEMINI XS, Molecular Devices, Inc., Sunnyvale, CA).
2.8. Statistical analysis Statistical evaluation was performed with the two-tailed Student’s t-test for unpaired values. Differences between the data were considered significant when P < 0.05. The data are represented as the mean S.E.M.
3. Results 3.1. Induction of apoptosis in Clk-1-deficient mouse embryos Clk-1-deficient mouse embryos survive in the expected Mendelian ratio until E8.5, with a slight developmental delay compared to their wild-type counterparts (Fig. 1 A and B), however, they fail to develop beyond E10.5 (E). Many condensed and fragmental nuclei were detected with haematoxylin and eosin (HE) staining in clk-1-deficient embryos at E10.5 (F). TUNEL staining confirmed that apoptotic cells were present in clk-1-deficient embryos (H and J), but not in wildtype embryos (G and I). These results indicated that apoptosis was more frequently induced in clk-1-deficient embryos than wild-type embryos at E10.5. On the other hand, only a few TUNEL-positive cells were observed in clk-1-deficient embryos as in wild-type embryos at E8.5 (C and D). In the process of apoptosis, procaspase-3 is cleaved to the activated form of caspase-3 (Thornberry and Lazebnik, 1998). To detect the activated caspase-3, whole cell lysates from clk-1deficient and wild-type embryos at E10.5 were examined by Western blot analysis, using a specific antibody to activated casapse-3. The activated caspase-3 was detected in the clk-1deficient cell lysates containing 10 or 25 mg of total protein (Fig. 2A, clk-1/), but not in the wild-type cell lysates containing 50 mg of total protein (Fig. 2A, clk-1+/+). In order to detect the externalization of phosphatidylserine on the plasma membrane as another characteristic of apoptosis (Martin et al., 1995), cells dissociated from embryos at E10.5 were stained with annexin V. The annexin V-positive cells that were stained in green were more frequently observed in clk-1deficient cells than in wild-type cells (Fig. 2B). Quantification indicated that the frequency of annexin V-positive cells was 39% in clk-1-deficient cells and 3% in wild-type cells, respectively (C). Statistical analysis indicates that the frequency of annexin V-positive cells is significantly higher in clk-1-deficient cells than in wild-type cells (P < 0.0001). 3.2. Mitochondria-mediated apoptosis in Clk-1-deficient mouse embryos Apoptosis is mediated via many signaling pathways in response to either intracellular or extracellular stimuli (Orrenius et al., 2003). One of these pathways is a
Fig. 1. TUNEL staining of clk-1-deficient mouse embryos. (A–D) Embryos of wild-type (clk-1+/+) and clk-1-deficient (clk-1/) mice at E8.5 were assayed by TUNEL staining (brown) and counterstained with haematoxylin (blue). Only a few TUNEL-positive cells (arrows) were detected in both clk-1+/+ and clk-1/ mice. (E) Embryos of clk-1+/+ and clk-1/ mice at E10.5. Embryos of clk-1/ mice showed a conspicuous developmental delay. (F) Condensed and fragmental nuclei (arrows) characteristic of apoptosis were observed in clk-1/ mouse embryos at E10.5 with HE staining. Embryos of clk-1+/+ (G and I) and clk-1/ (H and J) mice at E10.5 were assayed by TUNEL staining and counterstained with haematoxylin. The areas of each square in (G) and (H) are enlarged into (I) and (J), respectively. Many apoptotic cells stained in brown were present in clk-1/ mice, but few were present in clk-1+/+ mice. Scale bars: 1 mm (E), 500 mm (A, B and G), 100 mm (C, D and H), 50 mm (F, I and J).
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Fig. 2. Induction of apoptosis in clk-1-deficient cells. (A) Western blot analysis of activated caspase-3. Whole embryonic lysates from clk-1+/+ or clk-1/ mice at E10.5 were resolved by SDS-PAGE and immunoblotted with anti-activated caspase-3 antibody followed by HRP-conjugated secondary antibody. The arrow indicates the activated form of caspase-3. (B) Annexin V staining of embryonic cells at E10.5. Cells dissociated from clk-1+/+ or clk-1/ mouse embryos were stained with annexin V-FITC for apoptotic cells (green), and Hoechst33342 for nuclei (blue), and were observed under a confocal laser scanning microscope. Scale bars: 50 mm. (C) Quantification of apoptotic cells in clk-1+/+ and clk-1/ embryos at E10.5. The number of annexin V-positive cells is expressed as a percentage of the total number of cells. Data represent the mean S.E.M. of five determinants. The total number of wild-type and clk-1-deficient cells scored was 2696 and 715, respectively. ***P < 0.0001.
mitochondria-mediated pathway by which cytochrome c is released from mitochondria into the cytoplasm as an initial step (Liu et al., 1996; Zhivotovsky et al., 1998). In order to determine whether mitochondria-mediated apoptosis occurs in clk-1deficient mouse embryos, we examined the subcellular localization of cytochrome c. Immunostaining for cytochrome c appeared rod-shaped in wild-type cells (Fig. 3A, clk-1+/+), indicating the mitochondrial localization of cytochrome c. By contrast, cytochrome c staining was distributed diffusely in the cytoplasm of clk-1-deficient cells (A, clk-1/), indicating the release of cytochrome c from the mitochondria into the cytoplasm. Clk-1deficient mice fail to synthesize CoQ that plays an essential role in the mitochondrial respiratory chain (Marbois and Clarke, 1996; Nakai et al., 2001). Therefore, we examined the mitochondrial respiratory function by staining the embryonic cells with JC-1, which appears red (Em. 580 nm) in cells with a high DCm and green (Em. 530 nm) in cells with a low DCm (Reers et al., 1991,
1995; Smiley et al., 1991), followed by determination of the emission ratio (580:530 nm) of JC-1 fluorescence. Fluorescent microscopy revealed that most mitochondria in clk-1-deficient cells were stained in green and the emission ratio was 0.51 0.09 (Fig. 3B and C). By contrast, most mitochondria in wild-type cells were stained in red and the emission ratio was 1.34 0.17. Statistical analysis of the difference between the emission ratios indicates that DCm is significantly lower in clk-1-deficient cells than in wild-type cells (P < 0.0001). ATP is the final product of mitochondrial oxidative phosphorylation, therefore we examined whether the decrease in DCm is accompanied by a reduction in cellular ATP levels in clk-1-deficient cells. We revealed that the ATP level in clk-1deficient cells was 48% of that in wild-type cells (Fig. 3D, P < 0.0001). The results suggested that the mitochondrial respiratory function was severely impaired in clk-1-deficient embryonic cells compared to wild-type cells.
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3.3. Effects of exogenous CoQ10 on mitochondrial function and apoptosis in Clk-1-deficient embryonic cells Since clk-1-deficient mice fail to synthesize CoQ (Nakai et al., 2001), we examined whether exogenous CoQ restores the impaired function of mitochondrial respiration in clk-1-deficent cells by treating clk-1-deficient cells with 25 mM of exogenous water-soluble CoQ10 (sCoQ10) for 5 h, then determining of DCm by JC-1 staining. Although the emission ratio of JC-1 fluorescence in wild-type cells did not change significantly after treatment with sCoQ10, that in clk-1-deficient cells increased from 0.68 0.01 to 0.86 0.04 (P< 0.0001), but it was still lower than that in wild-type cells not treated with sCoQ10 (Fig. 4A). Similarly, a 5-h-incubation with sCoQ10 did not affect ATP levels in wild-type cells but significantly increased them from 65% to 77% in clk-1-deficient cells (Fig. 4B, P = 0.026). However, the ATP level in clk-1-deficient cells was still lower than that in wild-type cells after treatment with sCoQ10 (B). The results indicated that the decreased DCm and ATP levels in clk-1-deficient cells were partially but significantly restored by exogenous sCoQ10. To address whether exogenous sCoQ10 suppresses the apoptosis induced in clk-1-deficient cells, the dissociated cells were stained with annexin V after treatment with or without 25 mM sCoQ10 for 5 h. Treatment with exogenous sCoQ10 significantly reduced the frequency of annexin V-positive cells from 38.3% to 18.0% in clk-1-deficient cells (P < 0.0001) as well as in wild-type cells (P < 0.05) (Fig. 4C). 4. Discussion Histological observations revealed many condensed and fragmental nuclei and TUNEL-positive cells to be present in clk-1-deficient mouse embryos at E10.5 but not at E8.5 (Fig. 1), suggesting that apoptosis was induced in the clk-1-deficient embryos between E8.5 and E10.5. Detection of activated caspase-3 and externalization of phosphatidylserine on the plasma membrane further confirmed the induction of massive apoptotic cell death in whole mouse clk-1-deficient embryos at E10.5. Apoptosis is induced via two different pathways (Riedl and Shi, 2004). The first is the intrinsic mitochondria-mediated pathway by which cytochrome c is released from mitochondria into the cytoplasm (Liu et al., 1996), concomitant with a reduction in the DCm and ATP levels (Goldstein et al., 2000; Eguchi et al., 1997; Leist et al., 1997). The second is the extrinsic receptor-mediated signaling pathway (Orrenius et al., 2003). The present results demonstrating the release of Fig. 3. Mitochondria-mediated apoptosis induced in clk-1-deficient cells. (A) Subcellular localization of cytochrome c. Cells dissociated from clk-1+/+ and clk-1/ mouse embryos at E10.5 were stained with anti-cytochrome c antibody (red) and stained with Hoechst33342 for the nuclei (blue). Cytochrome c is distributed diffusely in the cytoplasm of clk-1-deficient cells. Clk-1/ cells show fragmented nuclei characteristic of apoptosis. Scale bars: 10 mm. (B) Cells from clk-1+/+ or clk-1/ mouse embryos at E10.5 were stained with JC-1 and Hoechst33342. Mitochondria with a low DCm are indicated in green (Em. 530) and those with high DCm are in red (Em. 580). Scale bars: 10 mm. (C)
Mitochondrial membrane potential was quantified by measuring the 580/ 530 nm ratio of JC-1 fluorescence intensity in 16 and 20 different fields of clk-1+/+ and clk-1/ cells, respectively. Data represent the mean S.E.M. ***P < 0.0001 (D) ATP levels in embryonic cells. Cells dissociated from clk1+/+ or clk-1/ mouse embryos at E10.5 were incubated with an ATP assay reagent, and the luminescence was measured with a luminometer. ATP levels are represented as a percentage of that in wild-type cells. Experiments were repeated three times, and the mean S.E.M. of five determinants in a representative experiment is given. ***P < 0.0001.
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Fig. 4. Effects of CoQ10 on mitochondrial membrane potential, ATP levels, and induction of apoptosis in clk-1-deficient cells. Dissociated cells from clk-1+/+ and clk-1/ embryos were cultured with or without 25 mM sCoQ10 for 5 h. Cells were stained with JC-1 and the 580:530 nm ratio of JC-1 fluorescence intensity was calculated to estimate mitochondrial membrane potential (A). Contents of ATP were determined as described in Section 2. ATP levels are represented as a percentage of that in wild-type cells without sCoQ10 (B). Apoptosis was assessed by annexin V staining as in Fig. 2C. Counted were more than five visual fields containing n > 150 cells for every experimental group. Results are the mean for five randomly selected fields, with a minimum of 150 cells scored per field (C). Each experiment was repeated two or three times and data represent the mean S.E.M. of five or six determinants in a representative experiment. *P < 0.05, **P < 0.001, ***P < 0.0001.
cytochrome c into the cytoplasm as well as the reduction in both the DCm and ATP level in clk-1-deficient cells (Fig. 3) suggest that the apoptosis induced in clk-1-deficient cells is mediated by the mitochondrial pathway. From these results we suggest that clk-1-deficiency induces mitochondria-mediated apoptosis in mouse embryonic cells. This is, however, inconsistent with the previous report that embryonic stem (ES) cells derived from clk-1-deficient mice were resistant to apoptosis in vitro (Liu et al., 2005). This discrepancy may be due to the difference of cell type used in each study, since ES cells have specific molecular features distinct from embryonic cells (Li et al., 1992; Meshorer et al., 2006; Boyer et al., 2006). Clk-1 encodes DMQ hydroxylase which catalyzes the biosynthesis of CoQ, an essential factor in oxidative phosphorylation (Stenmark et al., 2001). Therefore, clk-1-
deficient mice fail to synthesize CoQ and accumulate DMQ, the precursor of CoQ (Nakai et al., 2001; Levavasseur et al., 2001). The physiological function of DMQ in mitochondrial respiration is controversial in clk-1-deficient model organisms. Some reports suggest that DMQ can function as an electron carrier in the respiratory chain in clk-1 mutant nematodes (Miyadera et al., 2001; Felkai et al., 1999) and that CoQ is not essential for mitochondrial respiration in mouse (Levavasseur et al., 2001). By contrast, other studies suggest that DMQ is not able to support mitochondrial respiration in clk-1-deficient yeasts and nematodes (Padilla et al., 2004; Kayser et al., 2004). Our studies are consistent with the latter. In our previous study, an aberrant morphology of mitochondria was demonstrated in clk1-deficient mouse embryos, which implied mitochondrial dysfunction (Nakai et al., 2001). The present study indicated
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that embryonic cells from clk-1-deficient mice at E10.5 were partly stained in red with JC-1 (Fig. 3B), suggesting that DMQ could transport electrons to a lesser extent than CoQ. However, the present results clearly indicated that both DCm and ATP levels were significantly reduced in clk-1-deficient cells (Fig. 3 C and D), suggesting that DMQ accumulated in clk-1-deficient cells (Levavasseur et al., 2001; Nakai et al., 2001) and induced mitochondrial dysfunction. In addition, exogenous sCoQ10 significantly rescued the mitochondrial dysfunction in clk-1deficient cells (Fig. 4A and B). These results suggested that a lack of CoQ10 due to clk-1-deficiency induced the mitochondrial respiratory dysfunction in mouse embryonic cells. The present study showed the induction of mitochondrial respiratory dysfunction and massive apoptosis in clk-1deficient mice. Additionally, exogenous sCoQ10 significantly restored mitochondrial function and prevented the induction of apoptosis in clk-1-deficient cells (Fig. 4). Therefore, the present study suggests that mitochondrial respiratory dysfunction results in the induction of apoptosis in mouse embryos. This assumption is strongly supported by the finding that respiratory chain deficiency makes cells more prone to apoptosis in mouse embryos depleted of mitochondrial transcription factor A (Tfam) (Wang et al., 2001). It is not clear why clk-1-deficient mice die at around E10.5. One possibility is that CLK-1 protein or its enzyme activity preserved in oocytes descended from a heterozygous mother is depleted around E10.5 in mouse embryos. Since clk-1-deficient mice were produced from matings between heterozygous male and female mice in the present study, clk-1-deficient zygotes contained substantial amounts of the CLK-1 enzyme derived from maternal mitochondria. It is reported to be no or limited mitochondrial biogenesis during early embryogenesis (Shoubridge and Wai, 2007). Therefore, depletion of maternal CLK-1 protein until E9.5 may induce mitochondrial dysfunction resulting in embryonic death in clk-1-deficient mice. Actually, we previously detected neither CoQ9 nor CoQ10 in clk-1deficient embryos at E9.5 (Nakai et al., 2001). Another possibility may be that mitochondrial respiration changes from being anaerobic to aerobic at E8.5–10.5 in mouse embryos. During mouse embryogenesis, the development of the heart and angiogenesis in the yolk sac are induced at E8.5–10.5 (Kaufman and Navaratnam, 1981; Kaufman, 1991), which are closely associated with the oxygen supply to embryos. In addition, the mitochondrial inner membrane matures in rat embryos at E10.5–12.5 (Shepard et al., 1998), which correspond to E8.5–10.5 in mouse embryos. All these studies indicate that mitochondrial respiration changes from an anaerobic to aerobic process at E8.5–10.5 in mouse embryos. A small amount of ATP is generated in the glycolytic pathway under anaerobic conditions. However, after mitochondrial maturation, a large amount of ATP is produced through oxidative phosphorylation, with consequences for embryonic growth and maturation. Whatever the possibility may be, mice in which oxidative phosphorylation was abolished died around E9.5, including mice with a disrupted gene encoding cytochrome c (Li et al., 2000) or mitochondrial Tfam (Larsson et al., 1998), and clk-1 as well (Nakai et al., 2001).
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In summary, we showed that apoptosis was induced in clk-1deficient mouse embryos during E8.5–10.5. In addition, exogenous sCoQ10 restored the reduction of DCm and ATP levels and prevented apoptosis in clk-1-deficient cells at E10.5. Taken together, these results suggest that clk-1-deficiency causes embryonic lethality at E10.5 by the induction of mitochondria-mediated apoptosis due to a lack of CoQ in mice. Acknowledgements We thank N. Oshima (GE Health Care Bioscience Co. Ltd.) for expert assistance with the IN Cell Analyzer 1000 and I. Yoshimura (Nisshin Pharma Inc., Japan) for generously supplying water-soluble CoQ10 and its placebo. We are grateful to W. Zhou (Tokyo Metropolitan Institute of Gerontology) for preparing the manuscript. This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (15603009, to MT), and from Comprehensive Research on Aging and Health (13030101, to TS). References Andersson, S.G., Zomorodipour, A., Andersson, J.O., et al., 1998. The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature 396, 133–140. Asaumi, S., Kuroyanagi, H., Seki, N., Shirasawa, T., 1999. Orthologues of the Caenorhabditis elegans longevity gene clk-1 in mouse and human. Genomics 58, 293–301. Boyer, L.A., Plath, K., Zeitlinger, J., et al., 2006. Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441, 349– 353. Eguchi, Y., Shimizu, S., Tsujimoto, Y., 1997. Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res. 57, 1835–1840. Ernster, L., Dallner, G., 1995. Biochemical, physiological and medical aspects of ubiquinone function. Biochim. Biophys. Acta 1271, 195–204. Ewbank, J.J., Barnes, T.M., Lakowski, B., Lussier, M., Bussey, H., Hekimi, S., 1997. Structural and functional conservation of the Caenorhabditis elegans timing gene clk-1. Science 275, 980–983. Felkai, S., Ewbank, J.J., Lemieux, J., Labbe, J.C., Brown, G.G., Hekimi, S., 1999. CLK-1 controls respiration, behavior and aging in the nematode Caenorhabditis elegans. EMBO J. 18, 1783–1792. Goldstein, J.C., Waterhouse, N.J., Juin, P., Evan, G.I., Green, D.R., 2000. The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nat. Cell Biol. 2, 156–162. Hinegardner, R.T., 1971. An improved fluorometric assay for DNA. Anal. Biochem. 39, 197–201. Jonassen, T., Larsen, P.L., Clarke, C.F., 2001. A dietary source of coenzyme Q is essential for growth of long-lived Caenorhabditis elegans clk-1 mutants. Proc. Natl. Acad. Sci. U.S.A. 98, 421–426. Jonassen, T., Marbois, B.N., Kim, L., et al., 1996. Isolation and sequencing of the rat Coq7 gene and the mapping of mouse Coq7 to chromosome 7. Arch. Biochem. Biophys. 330, 285–289. Kaufman, M.H., 1991. Histochemical identification of primordial germ cells and differentiation of the gonads in homozygous tetraploid mouse embryos. J. Anat. 179, 169–181. Kaufman, M.H., Navaratnam, V., 1981. Early differentiation of the heart in mouse embryos. J. Anat. 133, 235–246. Kayser, E.B., Sedensky, M.M., Morgan, P.G., Hoppel, C.L., 2004. Mitochondrial oxidative phosphorylation is defective in the long-lived mutant clk-1. J. Biol. Chem. 279, 54479–54486. Lakowski, B., Hekimi, S., 1996. Determination of life-span in Caenorhabditis elegans by four clock genes. Science 272, 1010–1013.
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Mechanisms of Ageing and Development 129 (2008) 291–298 www.elsevier.com/locate/mechagedev
Clk-1 deficiency induces apoptosis associated with mitochondrial dysfunction in mouse embryos Mayumi Takahashi a, Takahiko Shimizu a, Eiko Moriizumi a, Takuji Shirasawa a,b,* a
Research Team for Molecular Biomarkers, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173-0015, Japan b Department of Aging Control Medicine, Juntendo University Graduate School of Medicine, Bunnkyou-ku, Tokyo 113-0033, Japan Received 14 August 2007; received in revised form 11 December 2007; accepted 27 January 2008 Available online 12 February 2008
Abstract Clk-1 gene encodes demethoxyubiquinone hydroxylase that catalyzes the production of coenzyme Q (CoQ) in mitochondria. Clk-1-deficient mice that lack CoQ fail to survive beyond the embryonic day 10.5 (E10.5). However, the relationship between the clk-1-deficiency and embryonic lethality remains unclear. We show in this study that TUNEL-positive cells are frequently observed in whole bodies of clk-1-deficient mouse embryos at E10.5. In addition, dissociated cells from the embryos exhibited characteristic features of apoptosis, such as externalization of phosphatidylserine on the plasma membrane, caspase-3 activation, and the release of cytochrome c from mitochondria into the cytoplasm, as the first sign of mitochondria-mediated apoptosis. In embryonic cells, the mitochondrial functions such as maintenance of the mitochondrial membrane potential and intracellular ATP level were impaired. Since exogenous CoQ10 rescued the mitochondrial dysfunction and suppressed apoptosis in clk-1-deficent cells, we propose that clk-1-deficency induces apoptosis associated with mitochondrial dysfunction due to a lack of CoQ, which may lead to embryonic lethality in mice around E10.5. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Clk-1; Apoptosis; Embryonic lethality; Coenzyme Q; Mitochondria; Mouse embryo
1. Introduction Clk-1 was initially reported in Caenorhabditis elegans as a gerontogene that is evolutionarily conserved among eukaryotes from yeast to human (Ewbank et al., 1997; Asaumi et al., 1999; Vajo et al., 1999; Takahashi et al., 2001; Jonassen et al., 1996) and in some bacteria as well (Stenmark et al., 2001; Andersson et al., 1998) and encodes demethoxyubiquinone (DMQ) hydroxylase that converts DMQ to 5-hydroxy-ubiquinone in the biosynthesis of coenzyme Q (CoQ)/ubiquinone (Marbois and Clarke, 1996). All the clk-1 mutants of yeasts, nematodes, and mice fail to synthesize CoQ, but instead accumulate DMQ (Jonassen et al., 2001; Levavasseur et al., 2001; Nakai et al., 2001). Among the functions of CoQ, the most pivotal one is to
* Corresponding author at: Department of Aging Control Medicine, Juntendo University Graduate School of Medicine, 3-3-10-201 Hongou, Bunnkyou-ku, Tokyo 113-0033, Japan. Tel.: +81 3 3814 1134; fax: +81 3 3814 1134. E-mail address: [email protected] (T. Shirasawa). 0047-6374/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2008.01.008
shuttle electrons from complex I or II to III in the mitochondrial respiratory chain, thereby leading to maintenance of mitochondrial membrane potential (DCm) and the production of ATP by oxidative phosphorylation (Ernster and Dallner, 1995; Turunen et al., 2004). Clk-1 mutants of C. elegans exhibit a long lifespan (Wong et al., 1995; Lakowski and Hekimi, 1996) when supplied with CoQ in their diet (Jonassen et al., 2001), but their development is arrested at the L2 larval stage when fed with mutant E. coli, deficient in CoQ (Jonassen et al., 2001). To investigate whether clk-1 plays a role in the embryonic development or determination of lifespan in mammals, we generated clk-1deficient mice (Nakai et al., 2001). Clk-1-deficient mice fail to survive beyond embryonic day 10.5 (E10.5) (Nakai et al., 2001; Levavasseur et al., 2001). However, the mechanism by which clk-1-deficiency leads to embryonic lethality remains to be elucidated. In this study, we examined this problem using clk-1deficient mouse embryos and cells isolated from the embryos just before death. A significant reduction in mitochondrial respiratory function and frequent mitochondria-mediated
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apoptosis were induced in the clk-1-deficient mouse embryos or cells. Since exogenous CoQ10 rescued the mitochondrial dysfunction and suppressed the induction of apoptosis, we suggest that clk-1 plays a crucial role in the maintenance of mitochondrial respiratory function and cell survival through the production of CoQ during the early development of mice. 2. Materials and methods 2.1. Animals Mice used in this study were F1 hybrids of the C57BL/6CrSlc strain (Nakai et al., 2001) and ICR strain (Japan SLC, Inc.). Animals were housed in specific pathogen-free facilities on a 12-h light/dark cycle and were fed ad libitum. All protocols for animal use and experiments were reviewed and approved by the Animal Care Committee of the Tokyo Metropolitan Institute of Gerontology.
2.2. Cell preparation and treatment Embryos at E10.5 were dissected from the yolk sac and amnion, and digested with 2.4 U/ml Dispase I (Roche, Switzerland) and 10 mg/ml DNase I (Sigma) for 30 min at 37 8C in a CO2 incubator. The digestion was stopped by adding serum-free COSMEDIUM 001 (COSMO BIO Co., Ltd.) containing 100 U/ml penicillin and 100 mg/ml streptomycin. Dissociated cells were collected by centrifugation at 700 g for 5 min (Beckmann, CS-6KR) and resuspended in COSMEDIUM. In some experiments, dissociated cells were treated with 25 mM water-soluble CoQ10 (sCoQ10, Aqua Q10L10, Nisshin Pharma Inc., Japan) or placebo (Nisshin Pharma Inc., Japan) at the same concentration of sCoQ10 dissolved in serum-free COSMEDIUM 001 for 5 h before each assay. Genotypes of mice or embryos were determined by PCR as described previously (Nakai et al., 2001).
2.3. Histological detection of apoptosis Mouse embryos at E8.5 and 10.5 were fixed in 20% (v/v) formalin, dehydrated, embedded in paraffin, and sectioned at 3 mm. TdT-mediated XdUTP nick end labeling (TUNEL) for the detection of apoptosis in embryos was performed using a TUNEL Label kit (Roche Molecular Biochemicals). Briefly, deparaffinized and rehydrated sections were incubated with proteinase K (20 mg/ml, Roche Molecular Biochemicals) for 60 min at 37 8C, rinsed twice with phosphate-buffered saline (PBS), and incubated in 0.3% H2O2-containing methanol for 10 min at room temperature to block the endogenous peroxidase (POD) activity. After being washed in PBS, sections were incubated with TUNEL reaction mixture containing terminal deoxynucleotidyl transferase in moisture overnight at 4 8C, washed again with PBS, and incubated with converter-POD solution for 1 h at room temperature. For the visualization of apoptotic signals, sections were incubated with 30 -diamino-benzidine (DAB) as a chromogen and counterstained with haematoxylin. The apoptosis in mouse embryo was quantified using an annexin V-FITC Apoptosis Detection kit (Sigma). Dissociated cells from embryos at E10.5 or cells treated with sCoQ10 were stained with 0.5 mg/ml annexin V-FITC and 5 ng/ml Hoechst33342 (Calbiochem) for nuclear counterstaining in annexinbinding buffer, followed by fixation in 20% (v/v) neutralized formalin containing 0.25 mM CaCl2 at room temperature for 10 min in the dark. Fluorescence images were recorded through bandpass filters of 420–480 and 505–530 nm, and the numbers of both annexin-positive and total cells were counted for five distinct fields in each of five wells, respectively using a IN Cell Analyzer 1000 (GE Healthcare Bio-Sciences Corp.). The same experiment was repeated three times. Annexin-positive cells are expressed as a percentage of the total number of nuclei.
2.4. Immunocytochemistry Cells dissociated from mouse embryo at E10.5 were cultured on glassbottom dishes (Matsunami Glass Ind. Ltd.) with serum-free COSMEDIUM in a
humidified atmosphere of 5% CO2 in air at 37 8C for 4 h and fixed in a solution of 50% (v/v) methanol/50% (v/v) acetone (20 8C) for 5 min on ice. After being incubated in blocking buffer (10% goat serum in PBS) for 30 min at room temperature, cells were incubated with anti-cytochrome c antibody (BD PharMingen) overnight at 4 8C, followed by Alexa Fluor 546-conjugated goat anti-mouse secondary antibody (Molecular Probes, Inc., Eugene, OR) for 1 h at room temperature. Nuclei were counterstained with 5 ng/ml Hoechst33342. Finally, the samples were embedded in non-fluorescence glycerol (Merck) diluted with PBS and observed under a confocal laser scanning microscope (Zeiss LSM5 PASCAL), equipped with a blue diode and a He/Ne laser with excitation at 405 and at 543 nm, respectively. Fluorescence images were recorded through a band pass filter at 420–480 nm and a long pass filter at 580 nm, respectively.
2.5. Western blot analysis Mouse embryos at E10.5 were homogenized with sonication in ice-cold lysis buffer (10 mM Tris–HCl, containing 150 mM NaCl, 1% SDS and protease inhibitor cocktail, CompleteTM (Roche)). Homogenates were centrifuged at 10,000 g for 10 min at 4 8C, and the concentration of protein in the supernatant was measured with a DC Protein Assay Kit (Bio-Rad, Hercules, CA). Proteins (10–50 mg/treatment) were resolved by 15% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Immobilon-PSQ, Bio-Rad). The membrane was blocked with 5% milk in TBS/Tween (Tris-buffered saline with 0.05% (v/v) Tween 20) for 1 h, and incubated with anti-activated caspase-3 (Cell Signaling Tech., Inc., Danver, MA) (1:1000 in 5% milk TBS/Tween) overnight at 4 8C. The blot was washed with TBS/Tween and incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000, Gibco BRL) for 1 h at room temperature. After washing, the immunoreactive protein was detected with an ECL system (GE Healthcare Bio-Sciences Corp.) and Luminoimage Analyzer LAS-3000 (Fuji, Tokyo, Japan).
2.6. Determination of mitochondrial membrane potential Mitochondrial membrane potential (DCm) was measured using 5,50 ,6,60 tetrachloro-1,10 ,3,30 -tetraethylbenzimidazole carbocyanideiodide (JC-1, Calbiochem), a cationic cyanine fluorescence dye incorporated into mitochondria in proportion to DCm (Reers et al., 1991, 1995; Smiley et al., 1991). A stock solution of JC-1 was prepared at 2 mg/ml in dimethyl sulfoxide. Dissociated embryonic cells at E10.5 or cells treated with sCoQ10 were stained with a mixture of 1 mg/ml JC-1 and 5 ng/ml Hoechst33342 in culture medium for 10 min in a CO2 incubator, washed three times with Hanks’ solution, and observed under a confocal laser scanning microscope (Carl Zeiss). Fluorescence images of monomers and J-aggregates were simultaneously captured through a 505–535 nm bandpass filter and a 580 nm long pass filter, respectively. The nuclear staining with Hoechst33342 was detected with a 420–480 nm bandpass filter after excitation with a blue diode laser at 405 nm. The ratio of absorbance at 580–530 nm (580/530 ratio) was calculated as the relative DCm value.
2.7. Measurement of ATP levels The ATP levels in cells were determined using a bioluminescence assay kit (Promega Corp., Madison, WI). Dissociated embryonic cells were immediately collected by centrifugation and suspended in Hanks’ solution. In some experiments to examine the effect of CoQ on ATP production, dissociated cells were cultured in a 96-well-plate containing COSMEDIUM with or without 25 mM sCoQ10 for 5 h in a CO2 incubator. After cultivation, the plate was centrifuged at 2000 rpm for 5 min (KUBOTA 7800, RS-96S, Japan) and medium was discarded until 10 ml was left in each well. Cell suspensions just after dissociation or cultured cells in wells were incubated with the same volume of ATP assay reagent to induce cell lysis on a shaker for 10 min at room temperature in the dark. After all samples were measured with a luminometer (Lumat LB9507, Berthold Technologies, Bad Wildbad, Germany), background luminescence without cells was subtracted from each value. ATP standard curves were run in all experiments with different concentrations of ATP, and calculations were made from the curve. DNA content was assessed according to the method of Hinegardner (1971) with small modifications in parallel to
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normalize the data. Briefly, cells were fixed with 100% ethanol for 5 min, airdried, and incubated with 0.4% 3,5-diaminobenzoic acid dihydrochloride (Sigma) for 45 min at 60 8C. The reaction was stopped with 2N HCl and fluorescence was measured in each sample or standard (Ex. 400 nm, Em. 530 nm) with a fluorometer (SPECTRA MAX GEMINI XS, Molecular Devices, Inc., Sunnyvale, CA).
2.8. Statistical analysis Statistical evaluation was performed with the two-tailed Student’s t-test for unpaired values. Differences between the data were considered significant when P < 0.05. The data are represented as the mean S.E.M.
3. Results 3.1. Induction of apoptosis in Clk-1-deficient mouse embryos Clk-1-deficient mouse embryos survive in the expected Mendelian ratio until E8.5, with a slight developmental delay compared to their wild-type counterparts (Fig. 1 A and B), however, they fail to develop beyond E10.5 (E). Many condensed and fragmental nuclei were detected with haematoxylin and eosin (HE) staining in clk-1-deficient embryos at E10.5 (F). TUNEL staining confirmed that apoptotic cells were present in clk-1-deficient embryos (H and J), but not in wildtype embryos (G and I). These results indicated that apoptosis was more frequently induced in clk-1-deficient embryos than wild-type embryos at E10.5. On the other hand, only a few TUNEL-positive cells were observed in clk-1-deficient embryos as in wild-type embryos at E8.5 (C and D). In the process of apoptosis, procaspase-3 is cleaved to the activated form of caspase-3 (Thornberry and Lazebnik, 1998). To detect the activated caspase-3, whole cell lysates from clk-1deficient and wild-type embryos at E10.5 were examined by Western blot analysis, using a specific antibody to activated casapse-3. The activated caspase-3 was detected in the clk-1deficient cell lysates containing 10 or 25 mg of total protein (Fig. 2A, clk-1/), but not in the wild-type cell lysates containing 50 mg of total protein (Fig. 2A, clk-1+/+). In order to detect the externalization of phosphatidylserine on the plasma membrane as another characteristic of apoptosis (Martin et al., 1995), cells dissociated from embryos at E10.5 were stained with annexin V. The annexin V-positive cells that were stained in green were more frequently observed in clk-1deficient cells than in wild-type cells (Fig. 2B). Quantification indicated that the frequency of annexin V-positive cells was 39% in clk-1-deficient cells and 3% in wild-type cells, respectively (C). Statistical analysis indicates that the frequency of annexin V-positive cells is significantly higher in clk-1-deficient cells than in wild-type cells (P < 0.0001). 3.2. Mitochondria-mediated apoptosis in Clk-1-deficient mouse embryos Apoptosis is mediated via many signaling pathways in response to either intracellular or extracellular stimuli (Orrenius et al., 2003). One of these pathways is a
Fig. 1. TUNEL staining of clk-1-deficient mouse embryos. (A–D) Embryos of wild-type (clk-1+/+) and clk-1-deficient (clk-1/) mice at E8.5 were assayed by TUNEL staining (brown) and counterstained with haematoxylin (blue). Only a few TUNEL-positive cells (arrows) were detected in both clk-1+/+ and clk-1/ mice. (E) Embryos of clk-1+/+ and clk-1/ mice at E10.5. Embryos of clk-1/ mice showed a conspicuous developmental delay. (F) Condensed and fragmental nuclei (arrows) characteristic of apoptosis were observed in clk-1/ mouse embryos at E10.5 with HE staining. Embryos of clk-1+/+ (G and I) and clk-1/ (H and J) mice at E10.5 were assayed by TUNEL staining and counterstained with haematoxylin. The areas of each square in (G) and (H) are enlarged into (I) and (J), respectively. Many apoptotic cells stained in brown were present in clk-1/ mice, but few were present in clk-1+/+ mice. Scale bars: 1 mm (E), 500 mm (A, B and G), 100 mm (C, D and H), 50 mm (F, I and J).
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Fig. 2. Induction of apoptosis in clk-1-deficient cells. (A) Western blot analysis of activated caspase-3. Whole embryonic lysates from clk-1+/+ or clk-1/ mice at E10.5 were resolved by SDS-PAGE and immunoblotted with anti-activated caspase-3 antibody followed by HRP-conjugated secondary antibody. The arrow indicates the activated form of caspase-3. (B) Annexin V staining of embryonic cells at E10.5. Cells dissociated from clk-1+/+ or clk-1/ mouse embryos were stained with annexin V-FITC for apoptotic cells (green), and Hoechst33342 for nuclei (blue), and were observed under a confocal laser scanning microscope. Scale bars: 50 mm. (C) Quantification of apoptotic cells in clk-1+/+ and clk-1/ embryos at E10.5. The number of annexin V-positive cells is expressed as a percentage of the total number of cells. Data represent the mean S.E.M. of five determinants. The total number of wild-type and clk-1-deficient cells scored was 2696 and 715, respectively. ***P < 0.0001.
mitochondria-mediated pathway by which cytochrome c is released from mitochondria into the cytoplasm as an initial step (Liu et al., 1996; Zhivotovsky et al., 1998). In order to determine whether mitochondria-mediated apoptosis occurs in clk-1deficient mouse embryos, we examined the subcellular localization of cytochrome c. Immunostaining for cytochrome c appeared rod-shaped in wild-type cells (Fig. 3A, clk-1+/+), indicating the mitochondrial localization of cytochrome c. By contrast, cytochrome c staining was distributed diffusely in the cytoplasm of clk-1-deficient cells (A, clk-1/), indicating the release of cytochrome c from the mitochondria into the cytoplasm. Clk-1deficient mice fail to synthesize CoQ that plays an essential role in the mitochondrial respiratory chain (Marbois and Clarke, 1996; Nakai et al., 2001). Therefore, we examined the mitochondrial respiratory function by staining the embryonic cells with JC-1, which appears red (Em. 580 nm) in cells with a high DCm and green (Em. 530 nm) in cells with a low DCm (Reers et al., 1991,
1995; Smiley et al., 1991), followed by determination of the emission ratio (580:530 nm) of JC-1 fluorescence. Fluorescent microscopy revealed that most mitochondria in clk-1-deficient cells were stained in green and the emission ratio was 0.51 0.09 (Fig. 3B and C). By contrast, most mitochondria in wild-type cells were stained in red and the emission ratio was 1.34 0.17. Statistical analysis of the difference between the emission ratios indicates that DCm is significantly lower in clk-1-deficient cells than in wild-type cells (P < 0.0001). ATP is the final product of mitochondrial oxidative phosphorylation, therefore we examined whether the decrease in DCm is accompanied by a reduction in cellular ATP levels in clk-1-deficient cells. We revealed that the ATP level in clk-1deficient cells was 48% of that in wild-type cells (Fig. 3D, P < 0.0001). The results suggested that the mitochondrial respiratory function was severely impaired in clk-1-deficient embryonic cells compared to wild-type cells.
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3.3. Effects of exogenous CoQ10 on mitochondrial function and apoptosis in Clk-1-deficient embryonic cells Since clk-1-deficient mice fail to synthesize CoQ (Nakai et al., 2001), we examined whether exogenous CoQ restores the impaired function of mitochondrial respiration in clk-1-deficent cells by treating clk-1-deficient cells with 25 mM of exogenous water-soluble CoQ10 (sCoQ10) for 5 h, then determining of DCm by JC-1 staining. Although the emission ratio of JC-1 fluorescence in wild-type cells did not change significantly after treatment with sCoQ10, that in clk-1-deficient cells increased from 0.68 0.01 to 0.86 0.04 (P< 0.0001), but it was still lower than that in wild-type cells not treated with sCoQ10 (Fig. 4A). Similarly, a 5-h-incubation with sCoQ10 did not affect ATP levels in wild-type cells but significantly increased them from 65% to 77% in clk-1-deficient cells (Fig. 4B, P = 0.026). However, the ATP level in clk-1-deficient cells was still lower than that in wild-type cells after treatment with sCoQ10 (B). The results indicated that the decreased DCm and ATP levels in clk-1-deficient cells were partially but significantly restored by exogenous sCoQ10. To address whether exogenous sCoQ10 suppresses the apoptosis induced in clk-1-deficient cells, the dissociated cells were stained with annexin V after treatment with or without 25 mM sCoQ10 for 5 h. Treatment with exogenous sCoQ10 significantly reduced the frequency of annexin V-positive cells from 38.3% to 18.0% in clk-1-deficient cells (P < 0.0001) as well as in wild-type cells (P < 0.05) (Fig. 4C). 4. Discussion Histological observations revealed many condensed and fragmental nuclei and TUNEL-positive cells to be present in clk-1-deficient mouse embryos at E10.5 but not at E8.5 (Fig. 1), suggesting that apoptosis was induced in the clk-1-deficient embryos between E8.5 and E10.5. Detection of activated caspase-3 and externalization of phosphatidylserine on the plasma membrane further confirmed the induction of massive apoptotic cell death in whole mouse clk-1-deficient embryos at E10.5. Apoptosis is induced via two different pathways (Riedl and Shi, 2004). The first is the intrinsic mitochondria-mediated pathway by which cytochrome c is released from mitochondria into the cytoplasm (Liu et al., 1996), concomitant with a reduction in the DCm and ATP levels (Goldstein et al., 2000; Eguchi et al., 1997; Leist et al., 1997). The second is the extrinsic receptor-mediated signaling pathway (Orrenius et al., 2003). The present results demonstrating the release of Fig. 3. Mitochondria-mediated apoptosis induced in clk-1-deficient cells. (A) Subcellular localization of cytochrome c. Cells dissociated from clk-1+/+ and clk-1/ mouse embryos at E10.5 were stained with anti-cytochrome c antibody (red) and stained with Hoechst33342 for the nuclei (blue). Cytochrome c is distributed diffusely in the cytoplasm of clk-1-deficient cells. Clk-1/ cells show fragmented nuclei characteristic of apoptosis. Scale bars: 10 mm. (B) Cells from clk-1+/+ or clk-1/ mouse embryos at E10.5 were stained with JC-1 and Hoechst33342. Mitochondria with a low DCm are indicated in green (Em. 530) and those with high DCm are in red (Em. 580). Scale bars: 10 mm. (C)
Mitochondrial membrane potential was quantified by measuring the 580/ 530 nm ratio of JC-1 fluorescence intensity in 16 and 20 different fields of clk-1+/+ and clk-1/ cells, respectively. Data represent the mean S.E.M. ***P < 0.0001 (D) ATP levels in embryonic cells. Cells dissociated from clk1+/+ or clk-1/ mouse embryos at E10.5 were incubated with an ATP assay reagent, and the luminescence was measured with a luminometer. ATP levels are represented as a percentage of that in wild-type cells. Experiments were repeated three times, and the mean S.E.M. of five determinants in a representative experiment is given. ***P < 0.0001.
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Fig. 4. Effects of CoQ10 on mitochondrial membrane potential, ATP levels, and induction of apoptosis in clk-1-deficient cells. Dissociated cells from clk-1+/+ and clk-1/ embryos were cultured with or without 25 mM sCoQ10 for 5 h. Cells were stained with JC-1 and the 580:530 nm ratio of JC-1 fluorescence intensity was calculated to estimate mitochondrial membrane potential (A). Contents of ATP were determined as described in Section 2. ATP levels are represented as a percentage of that in wild-type cells without sCoQ10 (B). Apoptosis was assessed by annexin V staining as in Fig. 2C. Counted were more than five visual fields containing n > 150 cells for every experimental group. Results are the mean for five randomly selected fields, with a minimum of 150 cells scored per field (C). Each experiment was repeated two or three times and data represent the mean S.E.M. of five or six determinants in a representative experiment. *P < 0.05, **P < 0.001, ***P < 0.0001.
cytochrome c into the cytoplasm as well as the reduction in both the DCm and ATP level in clk-1-deficient cells (Fig. 3) suggest that the apoptosis induced in clk-1-deficient cells is mediated by the mitochondrial pathway. From these results we suggest that clk-1-deficiency induces mitochondria-mediated apoptosis in mouse embryonic cells. This is, however, inconsistent with the previous report that embryonic stem (ES) cells derived from clk-1-deficient mice were resistant to apoptosis in vitro (Liu et al., 2005). This discrepancy may be due to the difference of cell type used in each study, since ES cells have specific molecular features distinct from embryonic cells (Li et al., 1992; Meshorer et al., 2006; Boyer et al., 2006). Clk-1 encodes DMQ hydroxylase which catalyzes the biosynthesis of CoQ, an essential factor in oxidative phosphorylation (Stenmark et al., 2001). Therefore, clk-1-
deficient mice fail to synthesize CoQ and accumulate DMQ, the precursor of CoQ (Nakai et al., 2001; Levavasseur et al., 2001). The physiological function of DMQ in mitochondrial respiration is controversial in clk-1-deficient model organisms. Some reports suggest that DMQ can function as an electron carrier in the respiratory chain in clk-1 mutant nematodes (Miyadera et al., 2001; Felkai et al., 1999) and that CoQ is not essential for mitochondrial respiration in mouse (Levavasseur et al., 2001). By contrast, other studies suggest that DMQ is not able to support mitochondrial respiration in clk-1-deficient yeasts and nematodes (Padilla et al., 2004; Kayser et al., 2004). Our studies are consistent with the latter. In our previous study, an aberrant morphology of mitochondria was demonstrated in clk1-deficient mouse embryos, which implied mitochondrial dysfunction (Nakai et al., 2001). The present study indicated
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that embryonic cells from clk-1-deficient mice at E10.5 were partly stained in red with JC-1 (Fig. 3B), suggesting that DMQ could transport electrons to a lesser extent than CoQ. However, the present results clearly indicated that both DCm and ATP levels were significantly reduced in clk-1-deficient cells (Fig. 3 C and D), suggesting that DMQ accumulated in clk-1-deficient cells (Levavasseur et al., 2001; Nakai et al., 2001) and induced mitochondrial dysfunction. In addition, exogenous sCoQ10 significantly rescued the mitochondrial dysfunction in clk-1deficient cells (Fig. 4A and B). These results suggested that a lack of CoQ10 due to clk-1-deficiency induced the mitochondrial respiratory dysfunction in mouse embryonic cells. The present study showed the induction of mitochondrial respiratory dysfunction and massive apoptosis in clk-1deficient mice. Additionally, exogenous sCoQ10 significantly restored mitochondrial function and prevented the induction of apoptosis in clk-1-deficient cells (Fig. 4). Therefore, the present study suggests that mitochondrial respiratory dysfunction results in the induction of apoptosis in mouse embryos. This assumption is strongly supported by the finding that respiratory chain deficiency makes cells more prone to apoptosis in mouse embryos depleted of mitochondrial transcription factor A (Tfam) (Wang et al., 2001). It is not clear why clk-1-deficient mice die at around E10.5. One possibility is that CLK-1 protein or its enzyme activity preserved in oocytes descended from a heterozygous mother is depleted around E10.5 in mouse embryos. Since clk-1-deficient mice were produced from matings between heterozygous male and female mice in the present study, clk-1-deficient zygotes contained substantial amounts of the CLK-1 enzyme derived from maternal mitochondria. It is reported to be no or limited mitochondrial biogenesis during early embryogenesis (Shoubridge and Wai, 2007). Therefore, depletion of maternal CLK-1 protein until E9.5 may induce mitochondrial dysfunction resulting in embryonic death in clk-1-deficient mice. Actually, we previously detected neither CoQ9 nor CoQ10 in clk-1deficient embryos at E9.5 (Nakai et al., 2001). Another possibility may be that mitochondrial respiration changes from being anaerobic to aerobic at E8.5–10.5 in mouse embryos. During mouse embryogenesis, the development of the heart and angiogenesis in the yolk sac are induced at E8.5–10.5 (Kaufman and Navaratnam, 1981; Kaufman, 1991), which are closely associated with the oxygen supply to embryos. In addition, the mitochondrial inner membrane matures in rat embryos at E10.5–12.5 (Shepard et al., 1998), which correspond to E8.5–10.5 in mouse embryos. All these studies indicate that mitochondrial respiration changes from an anaerobic to aerobic process at E8.5–10.5 in mouse embryos. A small amount of ATP is generated in the glycolytic pathway under anaerobic conditions. However, after mitochondrial maturation, a large amount of ATP is produced through oxidative phosphorylation, with consequences for embryonic growth and maturation. Whatever the possibility may be, mice in which oxidative phosphorylation was abolished died around E9.5, including mice with a disrupted gene encoding cytochrome c (Li et al., 2000) or mitochondrial Tfam (Larsson et al., 1998), and clk-1 as well (Nakai et al., 2001).
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