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Present address of K. Kamino is Division of Psychiatry and Behavioral Proteomics, Department of Post-Genomics and Diseases, Osaka University Graduate School of Medicine, 2–2, D3, Suita, 565–0871 Japan.
Department of Biochemistry and Cell Biology, Institute of Gerontology, Nippon Medical School, Kawasaki, Japan
Address correspondence and reprint requests to S. Ohta, Department of Biochemistry and Cell Biology, Institute of Gerontology, Nippon Medical School, 1–396 Kosugi-cho, Nakahara-ku, Kawasaki, 211–8533 Japan. E-mail: email@example.com
Mitochondrial aldehyde dehydrogenase 2 (ALDH2) plays a major role in acetaldehyde detoxification. The alcohol sensitivity is associated with a genetic deficiency of ALDH2. We have previously reported that this deficiency influences the risk for late-onset Alzheimer's disease. However, the biological effects of the deficiency on neuronal cells are poorly understood. Thus, we obtained ALDH2-deficient cell lines by introducing mouse mutant Aldh2 cDNA into PC12 cells. The mutant ALDH2 repressed mitochondrial ALDH activity in a dominant negative fashion, but not cytosolic activity. The resultant ALDH2-deficient transfectants were highly vulnerable to exogenous 4-hydroxy-2-nonenal, an aldehyde derivative generated by the reaction of superoxide with unsaturated fatty acid. In addition, the ALDH2-deficient transfectants were sensitive to oxidative insult induced by antimycin A, accompanied by an accumulation of proteins modified with 4-hydroxy-2-nonenal. Thus, these findings suggest that mitochondrial ALDH2 functions as a protector against oxidative stress.
Late-onset Alzheimer's disease (AD) is a complex disease caused by multiple genetic and environmental factors; physiological, medical, nutritional, and psychological. Recently, we have reported that the deficiency of mitochondrial aldehyde dehydrogenase (ALDH), ALDH2, is a risk factor for late-onset AD, synergistically acting with the ε4 allele of the apolipoprotein E gene (APOE-ε4; Kamino et al. 2000). However, the molecular mechanisms underlying the effects of the deficiency on neuronal function remain to be elucidated.
ALDH2 metabolizes acetaldehyde produced from ethanol into acetate and plays a major role in the oxidation of acetaldehyde in vivo (Bosron and Li 1986). A mutant allele, ALDH2*2, has a single point mutation (G/A) in exon 12 of the active ALDH2*1 gene and is confined to Asians (Yoshida et al. 1984). The mutation results in a substitution of glutamic acid 487 with lysine (E487K), acting in a dominant negative fashion (Crabb et al. 1989; Singh et al. 1989; Xiao et al. 1996). Individuals with the ALDH2*2 allele exhibit the alcohol flushing syndrome, attributable to elevated blood acetaldehyde (Goedde et al. 1979; Crabb 1990). The ALDH2*2 allele has been also reported to affect the metabolism of other aldehydes such as benzaldehyde, which is a metabolite of toluene (Kawamoto et al. 1994), and chloroacetaldehyde, which is generated during the metabolism of vinyl chloride (Farres et al. 1994; Yokoyama et al. 1996).
Oxidative stress and lipid peroxidation caused by reactive oxygen species (ROS) are reported to play an important role in the pathogenesis of neurodegenerative diseases, including AD (Lovell et al. 1997; Mark et al. 1997), Parkinson's disease (PD; Dexter et al. 1994), amyotrophic lateral sclerosis (Ferrante et al. 1997; Pedersen et al. 1998), and cerebral ischemia (for review, Chan 2001). A major ROS is mitochondrially derived superoxide anion radical, which attacks polyunsaturated fatty acids leading to membrane lipid peroxidation, thereby generating reactive aldehydes including 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde. A strong electrophile, 4-HNE, has the ability to readily adduct cellular proteins, which may damage the proteins by interacting with lysine, histidine, serine, and cysteine residues (Uchida and Stadtman 1992). Additionally, the high reactivity of 4-HNE makes it an ideal substrate for glutathione conjugation, resulting in depletion of cellular glutathione and disruption of the cellular redox status (Liu et al. 2000). Alternatively, 4-HNE inhibits Na+,K+-ATPase activity (Siems et al. 1996), presumably by directly binding the enzyme, and may give rise to neuronal damage. Evidence supports the neurotoxicity of 4-HNE, which results in injury and neuronal cell death through both apoptosis and necrosis (Kruman and Mattson 1999). Indeed, immunohistochemical analyses of post-mortem brain tissue from AD and PD patients revealed increased protein modification by 4-HNE in association with the neurodegenerative process (Yoritaka et al. 1996; Montine et al. 1997; Sayre et al. 1997).
We hypothesized that ALDH2 is involved in antioxidant defense and its deficiency enhances oxidative stress. As oxidative stress including ROS induced by β-amyloid follows the accumulation of 4-HNE (Keller et al. 1997), the deficiency of ALDH2 in neurons might influence the metabolism of 4-HNE. To verify this hypothesis, we obtained ALDH2-deficiect PC12 cells by transfection with a dominant negative form of the mouse Aldh2 gene, and found that ALDH2-deficient transfectants exhibited increased vulnerability to treatment with 4-HNE. The transfectants also had a decreased resistance to oxidative insult, caused by antimycin A, accompanied by an accumulation of proteins modified with 4-HNE. These findings suggest that mitochondrial ALDH2 functions as a protector against oxidative stress.
Materials and methods
Plasmid construction, cell culture, and transfection
A mouse wild-type Aldh2 cDNA was cloned from a mouse brain cDNA library (Life Technologies, Rockville, MD, USA) by PCR with a pair of primers, 5′-GTTCAGTTCGGGTCAGTTAAGCTCC and 5′-CAGTGTGTGTGGCGGTTTTTCTCA. To construct a mutant mouse Aldh2 gene (Aldh2*2), codon Glu506 (GAA) was substituted with Lys (AAA) by a two-step PCR mutagenesis method. For expression of the wild-type or mutant gene, each XhoI–BamHI fragment was inserted into pCAGGS (Niwa et al. 1991) digested with XhoI and BglII. Transfectant clones with the empty vector, the wild-type or mutant gene were examined by quantitative reverse transcriptase-PCR with a TaqMan real time PCR (7700; Applied Biosystems, Foster City, CA, USA). The PCR primers were selected to cover fragments from the vector to the insert. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Life Technologies) containing 10% fetal bovine serum and 5% horse serum on a collagen-coated dish (Iwaki, Tokyo, Japan). Constructed plasmids were cointroduced with pST-neoB (geneticin-resistant; Asoh et al. 2000) into PC12 cells with Effectene transfection regent (Qiagen, Valencia, CA, USA). The cells were cultured in microplates in the presence of geneticin and at the same time, cloned by the limited dilution method.
Cells were collected with ice-cold phosphate-buffered saline (PBS) and their wet weight was determined. A 16.7% (w/vol) cell suspension with 20 mm phosphate buffer (pH 7.4) containing 250 mm sucrose was prepared and the cytosolic and mitochondrial subcellular fractions were obtained by differential centrifugation as described previously (Yasukawa et al. 2001). In brief, cells were homogenized and centrifuged at 900 g for 5 min to collect supernatant. The obtained supernatant was further centrifuged at 10 000 g for 10 min to collect the precipitated mitochondria, and the remaining supernantant was further centrifuged at 100 000 g for 1 h to collect the cytosolic fraction containing the cytosolic ALDH activity. The mitochondrial fraction was sonicated and centrifuged at 100 000 g for 1 h to collect the mitochondrial ALDH activity. The protein concentration was determined with BCA protein assay reagent (Pierce, Rockford, IL, USA). ALDH activity was measured using the spectrophotometric assay, which described previously (Siew et al. 1976), with slight modification. In brief, the incubation mixture consisted of 20 mm phosphate buffer (pH 7.4), 1 mm NAD+ and 5 mm acetaldehyde, propionaldehyde or decylaldehyde as a substrate in a total volume of 100 µL. The reaction was initiated by addition of the substrate and incubated at 25°C for 10–30 min. Under these conditions, ALDH activity increased linearly with time at least until 30 min and was calculated as nmol NADH/min/mg protein.
4-HNE and antimycin A treatment
Stock solutions of 4-HNE (Calbiochem, San Diego, CA, USA) and antimycin A (Sigma, St Louis, MO, USA) were prepared in ethanol and then, just before use, diluted 1000-fold to achieve the working concentration in DMEM containing 1% fetal bovine serum. Corresponding amounts of ethanol were added to the control cultures. Before addition, cells were washed once with Hank's balanced salt solution. Cells were observed under a phase-contrast microscope (× 200) and viable cells were enumerated by the trypan blue exclusion method. PC12 transfectants with a smooth triangular soma were alive.
Determination of cellular oxidative stress
This measurement of cell oxidation is based on the oxidation of non-fluorescent 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Molecular Probes, Eugene, OR, USA) to a fluorescent product in a peroxidase-mediated reaction. After a wash with Hank's balanced salt solution, cells were incubated in DMEM with 1% fetal bovine serum containing 5 µm DCFH-DA and antimycin A (10 µg/mL). Four hours after treatment with or without antimycin A, cells were washed once with PBS, harvested, and analyzed by flow cytometry (EPICS ELITE ESP, Coulter, Hialeah, FL, USA) using excitation and emission filters of 488 and 525 nm.
Determination of intracellular 4-HNE levels
Immunocytochemistry was performed to measure 4-HNE generation in PC12 transfectants exposed to antimycin A by the method described previously (Ohsawa et al. 1999). Cultures of cells were prepared on 4-well plastic dishes (SonicSeal Slide; Nalge Nunc, Rochester, NY, USA) and treated with antimycin A or 4-HNE for 24 h with DMEM with 1% fetal bovine serum. Then, cells were rinsed with PBS and fixed in 4% paraformaldehyde in PBS for 30 min. After a wash with PBS, the cells were incubated for 30 min in 0.2% Triton X-100, 30 min in a blocking buffer (3% bovine serum albumin and 3% goat serum in PBS), and afterwards overnight at 4°C in a blocking buffer containing monoclonal anti4-HNE antibody (HNEJ-2, 10 µg/mL, purchased from Nikken Foods Co., Shizuoka, Japan). After an incubation with another wash with PBS, cells were incubated in a blocking buffer containing BODIPY FL goat anti-mouse IgG (1 : 500, Molecular Probes) for 1 h and imaged by confocal scanning microscopy using excitation and emission filters of 488 and 510 nm, respectively. Average pixel intensity was measured in each cell and expressed in relative units of fluorescence.
Characterization of PC12 cells transfected with a dominant negative form of the mouse Aldh2 gene
To elucidate the effect of the deficiency in ALDH2 on cell fate, we introduced a dominant negative form of the mouse Aldh2 gene (Aldh2*2) into PC12 cells. A nucleotide substitution in the human ALDH2 gene (ALDH2*2) is known to cause a deficiency of ALDH2 activity (Yoshida et al. 1984). Then, the Aldh2*2 gene was constructed by the substitution of Glu with Lys at the 14th last codon of the mouse wild-type Aldh2 gene. PC12 cells were stably transfected with a control vector and expression plasmids for wild-type Aldh2 and Aldh2*2 genes, respectively. The expression of each gene in the transfected cells was confirmed by quantitative reverse transcription-PCR. K6 and K11 were transfectants expressing Aldh2*2, while E was expressing Aldh2*1 (wild-type) in a similar extent with K6 and K11. A comparison of the mitochondrial ALDH activity in each transfectant is shown in Fig. 1(a). As expected, the activities of transfectants with the Aldh2*2 gene (K6, K11) were reduced compared to those of the parent cells (PC12), control transfectants (V), and the cells with the wild-type Aldh2 gene (E). On the other hand, the cytoslic ALDH-activity in each transfectant did not differ (Fig. 1b). Thus, the product of the Aldh2*2 gene specifically inhibited the ALDH2 activity of mitochondria. The transfection did not affect cell growth, or cell differentiation with nerve growth factor (data not shown). Cell survival rates after withdrawal of the serum from the medium were independent of the Aldh2*2 gene (Fig. 1c), indicating no effect by the expression of the gene on the cell viability under conventional culture conditions.
Sensitivity to 4-HNE-induced cell death in ALDH2-deficient PC12 cells
It has been reported that 4-HNE, an aldehydic product of lipid peroxidation, induces apoptosis in PC12 cells (Kruman et al. 1997), and that ALDH2 catalyzes the oxidation of 4-HNE to 4-hydroxy-2-nonenoic acid (Mitchell and Petersen 1987). Therefore, we examined the toxic effect of 4-HNE and found that an exposure to 4-HNE resulted in a more rapid decrease of viable cells in the ALDH2-deficient population than other control cells. Treatment with 10 µm 4-HNE resulted in the appearance of round cells, which progressively appeared at about 2 h post-treatment. The transfectants and controls were not distinguished in the extents. However, after 24 h, the ALDH2-deficient cells (K6 and K11) detached from the dish and aggregated (Fig. 2a), whereas the control cells (PC12, V and E) recovered a normal morphology. At that time, the percentage of living ALDH2-deficient cells (K6 and K11) was 36.7% and 35.1%, whereas that of control cells (PC12, V and E) was 98.6%, 85.3%, and 101.7%, respectively. Time-course study revealed that a day after exposure to 10 µm 4-HNE, the survival of ALDH2-deficient cells decreased rapidly, whereas that of control cells decreased gradually (Fig. 2b). The sensitivity of ALDH2-deficient cells to 4-HNE was dose-dependent (Fig. 2c). Treatment with 5 µm 4-HNE also significantly decreased the survival of the ALDH2-deficient cells. These findings clearly show that ALDH2-deficient cells are less resistant to exogenous 4-HNE.
Rapid cell death in ALDH2-deficient PC12 cells induced by antimycin A
Partial inhibition of the mitochondrial electron transport at complex III (cytochrome c reductase) by low concentrations of antimycin A induces the production of ROS and cell death (Turrens et al. 1985; Asoh et al. 1996). To investigate the effect of ALDH2 deficiency on cell vulnerability induced by the oxidative stress, at first we examined the cellular toxicity of antimycin A in the ALDH2-deficient and parental cells of PC12. Exposure of the ALDH2-deficient cells to antimycin A (10 µg/mL) resulted in a rapid loss of viable cells, whereas that of wild-type Aldh2-expressing cells and culture control cells did not (Fig. 3a). A day after the exposure to antimycin A, about 50% of K6 and K11 cells died, while more than 70% of other transfectants and parental PC12 cells were alive (Fig. 3b). The sensitivity of ALDH2-deficient cells to antimycin A was dose-dependent (Fig. 3c), suggesting that antimycin in this range contributes to the superoxide production.
Accumulation of 4-HNE in ALDH2-deficient PC12 cells by ROS
It has been reported that oxidative insults induce an accumulation of 4-HNE in PC12 to levels capable of inducing cell death (Kruman et al. 1997). Then, we examined whether a higher sensitivity of the ALDH2*2 transfectants to oxidative insults causes the accumulation of aldehyde products. To determine ROS levels after treatment with antimycin A, we used DCFH-DA which permeates into cells and interacts with intracellular ROS to generate a fluorescent dye. As shown in Fig. 4(a), a 4-h exposure to antimycin A induced fluorescence in each transfectant, indicating that antimycin A induces the generation of ROS in PC12 cells the same as its transfectants. Increases in ROS did not depend upon the type of transfectant. Then, we examined whether the accumulation of 4-HNE induced by the ROS differed between the ALDH2-deficient and -active cells. The accumulation after the exposure to antimycin A was measured with an anti4-HNE antibody in immunocytochemical assays. A day after treatment with antimycin A (3 or 10 µg/mL), cellular 4-HNE immunoreactivity increased only in ALDH2-deficient cells, K6 and K11, and not in control cells (Figs 4b and c). On the other hand, exogenous 1 µm 4-HNE induced immunoreactivity in all cells examined. These results strongly suggest that the ALDH2 deficiency caused the intracellular accumulation of 4-HNE, resulting in cell death.
ALDH2 plays a major role in the oxidation of acetaldehyde in vivo. Its low Km facilitates the rapid clearance of acetaldehyde following the administration of alcohol and a deficiency of ALDH2 results in an ethanol-related sensitive response, attributable to elevated blood acetaldehyde. Several reports have suggested that an increase in the acetaldehyde concentration as a risk for diabetes (Suzuki et al. 1996a, 1996b), cancer (Yokoyama et al. 1998), and hypertension (Itoh et al. 1997) is associated with ALDH2*2. Instead, this study proposes that ALDH2 could contribute to the pathogenesis of various geriatric diseases by an alternative pathway, which is the detoxification of cytotoxic products of lipid peroxidation.
Studies have revealed that 4-HNE, an aldehydic product of membrane lipid peroxidation, is a key mediator of neuronal cell death induced by oxidative insults involving β-amyloid and Fe2+ (Mark et al. 1997), and that the amount of protein modified with 4-HNE increases in AD brain. Specific events associated with 4-HNE-mediated cytotoxicity include inhibition of protein synthesis, neutrophil chemotaxis, induction of chromosomal aberrations and micronucleation, and formation of DNA adducts (for review see Comporti 1998). In neurons, the mechanism whereby 4-HNE increases its vulnerability to excitotoxicity may involve covalent modifications of membrane transporters, resulting in impairment of their function and disruption of calcium homeostasis (Kruman and Mattson 1999). Therefore, rapid detoxification of 4-HNE is important to cell survival. Oxidative, reductive, and conjugative metabolic pathways function during elimination of 4-HNE (Hartley et al. 1995). It was reported that the oxidative metabolite of 4-hydroxy-2-nonenoic acid, produced by ALDH, appeared rapidly and was exported into extracellular medium (Tjalkens et al. 1999). Thus, it is possible that the ALDH activity is primarily responsible for the elimination of 4-HNE.
Then, we, in this study, hypothesized that the deficiency of ALDH2 in neurons should decrease the capacity to metabolize 4-HNE, resulting in both an increase in cellular 4-HNE and neuronal cell death. Indeed, the present findings indicate that the ALDH2 deficiency in PC12 cells increases cell death after treatment with 4-HNE (Fig. 2). Interestingly, this finding indicates that, in spite of the much stronger activity of cytosolic than mictochondrial ALDHs, mitochondrial ALDH2 contributes most to the detoxification of exogenous 4-HNE. Furthermore, ALDH2-deficient cells accumulated proteins modified with 4-HNE, which may be generated by the reaction of unsaturated fatty acids with superoxide anion effused from complex III (Fig. 4). The concentration of antimycin A used to effuse the superoxide anion from complex III was low enough not to influence the oxidative phosphorylation (Schulze-Osthoff et al. 1992). Thus, it is unlikely that the reduction of ATP directly caused the cell death of the Aldh2*2 transfectants treated with antimycin A. Even if the ATP production is affected by antimycin A, the decrease itself was not fatal to cell death. Instead, these results strongly suggest that the enhanced accumulation of toxic acetaldehyde or aldehyde derivatives such as 4-HNE in the ALDH2-deficient cells induced the cell death. It is still possible that the decrease of the ATP production enhanced the sensitivity to the aldehydes. Taken together, it is concluded that mitochondrial ALDH2 is primarily responsible for the detoxification regardless of whether the 4-HNE is exogenous or endogenous.
Sixteen genes with distinct chromosomal locations have been identified in the human ALDH gene family. ALDHs have a similar primary structure and catalyze the oxidation of a wide spectrum of endogenous and exogenous aliphatic and aromatic aldehydes. Among members of the ALDH family, the ALDH2 gene is highly transcribed in the brain (Stewart et al. 1996). This study showed that PC12 transfectants with the Aldh2*2 gene, which were vulnerable to 4-HNE treatment, decreased mitochondrial ALDH activity, but not cytosolic activity (Fig. 1). Thus, in spite of the higher ALDH activity of the cytosolic fraction, mitochondrial ALDH2 would play a major role in the clearance of cytotoxic aldehydes derived from peroxides in neurons.
A majority of ROS are produced in mitochondria, resulting in peroxidation of the mitochondrial membrane. Because the mitochondrial inner membrane is rich in polyunsaturated fatty acids such as cardiolipin, reactive aldehydes including 4-HNE would be easily derived from the peroxidated polyunsaturated fatty acids. Thus, rapid elimination of 4-HNE is necessary in mitochondria. Recent findings of Reichard et al. (2000) support our explanations. They compared the rate of metabolism in stellate cells derived from normal and cirrhotic rat livers, and found that the greater rate of elimination of 4-HNE in cells from cirrhotic rat livers was attributable to higher mitochondrial ALDH activity.
It has been shown that AD patients homozygous for APOE-ε4 have greater 4-HNE adduct immunoreactivity associated with neurofibrillary tangles than AD patients with other APOE genotypes (Montine et al. 1997). Pedersen et al. (2000) reported an APOE isoform-specific modification with 4-HNE. Studies of the interactions of APOE proteins with 4-HNE showed that the isoforms differ in the amount of 4-HNE they can bind, with the order ε2 > ε3 > ε4. This correlated with the differential ability of APOE isoforms to protect against apoptosis induced by 4-HNE in cultured neurons. Our case–control study has revealed that ALDH2 deficiency is a risk factor for late-onset AD in a Japanese population, synergistically acting with APOE-ε4 (Kamino et al. 2000). When compared to carriers of the APOE-ε3/ε3 genotype, the risks for late-onset AD in Japanese subjects with the APOE-ε4 allele are twice those in Caucasian subjects (Haines et al. 1996). The increased risks can partly be explained by the effect of the ALDH2*2 allele, as this allele is very rare in populations except Asians. Therefore, we suggest the possibility that an enhancement of 4-HNE accumulation in AD brain by ALDH2 deficiency and a weaker activity of APOE-ε4 to protect against neuronal cell death induced by 4-HNE synergistcally act on late-onset AD. Nevertheless, Japanese patients with AD are less numerous than Caucasians, indicating that the other risks would overcome the risk of the ALDH2 deficiency. Recently, Picklo et al. (2001) reported that ALDH activity was significantly increased in the temporal cortex of AD patients. The results suggest that increased ALDH activity is a protective response to AD.
Finally, our results suggest that mitochondrial ALDH2 functions as a protector against oxidative stress. Thus, the metabolism of aldehyde including ALDH2 could be a preventive and therapeutic target in AD and other neurodegenerative disorders.