Induction of Bcl-2 and Bax was related to hyperphosphorylation of tau and neuronal death induced by okadaic acid in rat brain.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 287A:1236 –1245 (2005) Induction of Bcl-2 and Bax Was Related to Hyperphosphorylation of Tau and Neuronal Death Induced by Okadaic Acid in Rat Brain LI-QIN CHEN, JIAN-SHE WEI, ZHI-NIAN LEI, LING-MEI ZHANG, YAN LIU, AND FENG-YAN SUN* National Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, Shanghai, China ABSTRACT Abnormal hyperphosphorylation of the cytoskeletal protein tau is a characteristic feature of neurodegeneration in Alzheimer’s disease (AD) brain. Okadaic acid (OA), a protein phosphatase inhibitor, induces neuronal death and hyperphosphorylation of tau. In the present study using a model of microinjection of OA into rat frontal cortex, we aimed to investigate if OA-induced hyperphosphorylation of tau and neuronal death are related to the expression of Bcl-2, an apoptosis inhibitor, or Bax, an apoptosis inducer. Immunohistochemistry and Western blot analysis showed that OA injection dose- and time-dependently induced the expression of Bcl-2 and Bax protein in the surrounding of OA injection areas, which were similar with that of AT8 immunostaining, a marker of hyperphosphorylated tau. However, the ratios of Bcl-2 over Bax had a negative relationship to the expression of AT8. Furthermore, double ﬂuorescent staining showed that AT8-positive neurons mainly costained with terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick-end labeling, a marker of DNA damage, indicating that tau hyperphosphorylation may be associated with DNA damage in the neurons of rat brain. In the areas more adjacent to the OA injection site, most neurons with AT8-positive staining showed vulnerability to OA toxicity and could be triple-stained with Bcl-2 and Bax or double-stained with Bcl-2. However, in the areas further from the OA injection site, neurons with few AT8-positive staining showed resistance to OA toxicity and only stained with Bcl-2, but not Bax. The results suggest that the ratios of Bcl-2 over Bax expression may have an effect on tau hyperphosphorylation and neuronal death following OA injection. © 2005 Wiley-Liss, Inc. Key words: okadaic acid; apoptosis; tau phosphorylation; Bcl-2; Bax Alzheimer’s disease (AD) is characterized by neuronal loss and two pathologic hallmarks, amyloid deposits and neuroﬁbrillary tangles (NFTs) (Vickers et al., 2000). It has been shown that the number of NFTs correlates closely with the degree of dementia (Arriagada et al., 1992). NFTs are composed of abnormally hyperphosphorylated microtubule-associated protein (MAP) tau (Arriagada et al., 1992). Tau, mainly residing in neuronal axons, is phosphorylated at very low level in normal adult brain (Iqbal et al., 2000). Abnormal hyperphosphorylation of tau has been considered as a key pathologic molecular change correlated with neurodegeneration in AD brain (Iqbal et al., 2000). Hyperphosphorylated tau fails to bind microtubules, leading to disassembly of microtubules, disruption Grant sponsor: National Key Project of Basic Science Research and Shanghai Metropolitan Fund for Research and Development; Grant number: G1999054007 and 04DZ00415. The ﬁrst 2 authors contributed equally to this paper. *Correspondence to: Feng-Yan Sun, National Laboratory of Medical Neurobiology, Shanghai Medical College of Fudan Uni- versity, 138 Yi Xue Yuan Road, Shanghai 200032, China, Fax: 86-21-64174579. E-mail: firstname.lastname@example.org Received 6 July 2004; Accepted 10 June 2005 DOI 10.1002/ar.a.20241 Published online 1 November 2005 in Wiley InterScience (www.interscience.wiley.com). © 2005 WILEY-LISS, INC. INDUCTION OF Bcl-2 AND Bax of axonal transport, and ultimately retrograde neurodegeneration (Iqbal et al., 2000). Regulation of tau phosphorylation by protein kinases (PKs) and protein phosphatases (PPs) maintains dynamic balance under normal physiological conditions. Dephosphorylative activity of PP is the prerequisite in vivo for keeping tau at low phosphorylated level and with biological functions (Kins et al., 2001). PP activity has been reported to reduce by about 30% in AD brain compared to control (Gong et al., 1993, 1995). It has also been elucidated that defect of PP activity mimics Alzheimer-like tau pathology, including aberrant phosphorylation, altered compartmentalization, and colocalization with ubiquitin (Kins et al., 2001). Okadaic acid (OA), an inhibitor of PP, has been a useful tool to study AD (Bialojan and Takai, 1988). Treatment neurons with OA in vitro (Arias et al., 1993; Merrick et al., 1997; Tanaka et al., 1998; Kim et al., 1999) or in vivo (Arendt et al., 1995, 1998; Nelson and Saper, 1996; Mudher and Perry, 1998) induced aberrant hyperphosphorylation of tau or neuronal death or both. Previous studies showed that apoptosis was involved in neuronal death induced by OA or other inhibitors of PP in vitro (Boe et al., 1991; Nuydens et al., 1998; Fladmark et al., 1999) and in vivo (Arendt et al., 1998). Bcl-2 and Bax, belonging to Bcl-2 family, are important apoptotic regulatory molecules. The two, with contrary functions, integrate signals transducted from extracellular or intracellular insults at mitochondrial level (Yang et al., 1997; Hengartner, 2000). Their ratio has been suggested to be able to decide the cell’s fate under certain conditions. The two molecules both have widespread neuronal expression in the central nervous system (CNS) (Krajewski et al., 1994; Merry and Korsmeyer, 1997). Previous studies have proposed that Bcl-2 and Bax play important roles in neuronal vulnerability and resistance to degeneration in many CNS diseases, including AD (Krajewski et al., 1995; Martin, 1999; Vila et al., 2001; Raghupathi et al., 2002). However, it is not well deﬁned if Bcl-2 and Bax are involved in the pathphysiological process of Alzheimer-like neuronal death associated with hyperphosphorylation of tau. In the present study, we ﬁrst aimed to demonstrate that OA could induce neurodegeneration associated with hyperphosphorylation of tau with a model of microinjection of OA into rat cerebral cortex. Furthermore, with the model we explored whether the expression of Bcl-2 or Bax proteins was associated with hyperphosphorylation of tau induced by OA. Hyperphosphorylation of tau was assayed by AT8 antibody recognizing the aberrant phosphorylation site at serine 202, threonine 205 of tau. MATERIALS AND METHODS Animals All animal experiments were approved by the Medical Experimental Animal Administrative Committee of Shanghai and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize animal suffering and to reduce the number of animals used. Male Sprague-Dawley rats from Experimental Animal Center of Chinese Academy of Science were used for this study. Animals were anesthetized with chloral hydrate. For immunochemistry and ﬂuorescence immunostaining, animals were transcardially perfused with 0.9% saline solution followed by ﬁxative (4% paraformaldehyde in 0.1 M 1237 phosphate buffer) after anesthetization with chloral hydrate. After perfusion, the brains were removed and postﬁxed in the same ﬁxative for 8 hr, then immersed in 20% and 30% sucrose solution in 0.1 M phosphate buffer until sinking. Coronal serial sections were cut at 30 m thickness on a freezing microtome and stored in a cytoprotectant solution at ⫺20°C. For Western blot analysis, the cortex tissues were quickly collected on ice, frozen immediately in dry ice, and kept at ⫺70°C. Application of Okadaic Acid Saline (vehicle) or saline containing OA (pH 7.4; Sigma) was injected into the right frontal cortex of rats using stereotaxic surgery (stereocoordinates: A, 1.20 mm for anterior-posterior; L, 1.90 mm for lateral; H, 2.20 mm for dorsoventral). Rats were injected with 0.5 L of saline or saline containing 10, 20, 50, or 100 ng OA and sacriﬁced at 1 day. For Western blot analysis, injection of 50 ng OA was omitted. In addition, rats were sacriﬁced at 3 hr, 6 hr, 12 hr, 1 day, 3 days, or 7 days after 20 ng OA injection. For Western blot analysis, 6-hr group was not set. The number of animals used was four in each dose group of doseresponse or each time point group of time-course analysis. Immunohistochemistry Three sections (Bregma 1.20 mm) from each rat brain were randomly selected for immunochemistry. Free-ﬂoating sections from each rat brain were ﬁxed in 4% paraformaldehyde for 10 min, followed by three rinses in 0.01 M phosphate-buffered saline (PBS). Endogenous peroxidase activity was quenched by exposing the sections to 0.3% H2O2 for 30 min. After blocking with 10% normal goat serum for 30 min at 37°C, the sections were incubated with a mouse monoclonal AT8 antibody (1:60; Innogenetics, Belgium), with a rabbit polyclonal antibody against Bcl-2 (1:60; Oncogene Science), or with a mouse monoclonal antibody against Bax (1:40; Oncogene Science) in 0.01 M PBS containing 1% normal goat serum and 0.3% Triton X-100 for 2 hr at 37°C and overnight at 4°C. The sections were then rinsed and incubated for 1 hr at 37°C with antimouse or biotinalated antirabbit IgG (1:200; ABC kit, Vector Lab) in 0.01 M PBS containing 1% normal goat serum and 0.3% Triton X-100. Subsequently, they were incubated for 1 hr at 37°C with avidin-biotin-horseradish peroxidase complex (1:200; ABC kit, Vector Lab) following rinses. Immunostaining was visualized with 0.05% diaminobenzidene (Sigma) as chromogen. Negative control sections received identical treatment except for exposure to the primary antibodies and showed no speciﬁc staining. Double Staining Sections (Bregma 1.20 mm) from rats sacriﬁced at 1 day after 20 ng OA injection were doubly stained for AT8 and terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick-end labeling (TUNEL). Following incubation with a mouse monoclonal AT8 antibody as mentioned above, free-ﬂoating sections were rinsed with 0.01 M PBS and incubated with antimouse IgGrhodamine (1:20; Roche) for 1 hr at 37°C and rinsed again. After that, the sections were incubated with 20 g/ml protease K in 10 mM Tris-HCl (pH 7.6) for 15 min at 37°C. After rinsing with 0.01 M PBS, the sections were incubated with 0.1% Triton X-100 in 0.1% sodium citrate for 15 min and rinsed again. Then the sections were incubated 1238 CHEN ET AL. with TUNEL reaction mixture (in situ cell death detection kit; Boehringer Mannheim, Germany) composed of 45 l TUNEL label solution conjugated with ﬂuorescein and 5 l TUNEL enzyme for 90 min at 37°C. Negative control section for TUNEL staining was incubated with 50 l TUNEL label solution replacing TUNEL reaction mixture. The ﬂuorescence signals were observed under confocal laser scanning microscopy (TCS NT; Leica, Germany). Triple Staining Sections (Bregma 1.20 mm) from rats sacriﬁced at 1 day after 20 ng OA injection were triply stained for AT8, Bcl-2, and Bax. Free-ﬂoating sections were incubated for 2 hr at 37°C and overnight at 4°C in a mixture of two primary antibodies raised in separate species: a rabbit polyclonal antibody against Bcl-2 (1:60) and a mouse monoclonal AT8 antibody (1:60). Sections were then rinsed three times with 0.01 M PBS for 5 min each time and incubated for 1 hr at 37°C with the secondary antibodies, an antirabbit IgG-rhodamine (1:20; Roche), and an antimouse IgG-ﬂuorescein isothiocyanate (FITC; 1:20; Vector Lab). After three rinses with 0.01 M PBS, sections were incubated with a mouse monoclonal antibody against Bax (1:40) for 2 hr at 37°C and overnight at 4°C. Thereafter, sections were incubated with the secondary antibody, an antimouse IgG-CY5 (1:100; Amersham) for 1 hr at 37°C. The ﬂuorescence signals were observed under the same microscopy mentioned in double staining. The images were captured from two kinds of areas. One was the area immediately surrounding the OA injection site and called the more adjacent area. The other was the region relatively far from the OA injection site and called the less adjacent area. The edges of the more adjacent area were 0.42 mm apart from the injection site. The less adjacent area was the region of frontal cortex excluding the more adjacent area. Cell Counting As to each rat brain, three sections (Bregma 1.20 mm) immunostained were used for cell counting. Bcl-2-positive cell counts in affected brain areas by OA injection were measured with the aid of the image processing and analysis system. Counting of AT8- and Bax-positive cells in affected brain areas by OA injection was done under 400⫻ light microscopy. All values were expressed as mean ⫾ SEM. For AT8, Bcl-2, and Bax immunostaining at each time point after 20 ng OA injection, data were statistically analyzed with unpaired Student’s t-test. For AT8, Bcl-2, and Bax immunostaining at 1 day after serial doses of OA injection, the amount was evaluated by one-way ANOVA, followed by unpaired Student’s t-test. Statistical signiﬁcance was set at P ⬍ 0.05. Western Blot Analysis The samples were homogenized in lysis buffer (50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 1 g/ml aprotinin, and 100 g/ml PMSF). Protein concentration was measured by a Bio-Rad protein assay. Each sample (80 g protein) was boiled at 100°C in SDS sample buffer for 5 min, electrophoresed on 15% SDS-PAGE gels, and then transferred to polyvinyldiﬂuoridine (PVDF) membrane (Bio-Rad). The membrane was incubated sequentially with rabbit polyclonal antibody against Bcl-2 (1:1,000; Santa Cruz Bio- technology), mouse monoclonal antibody against Bax (1: 1,000; Santa Cruz Biotechnology), and mouse monoclonal antibody against β–isoform of actin (1:1,000; Sigma) overnight at 4°C. The membrane was washed with TBS containing 0.1% Tween 20, incubated with horseradish peroxidase-conjugated goat antirabbit IgG (1:1,000; Santa Cruz Biotechnology) and goat antimouse IgG (1:1,000; Santa Cruz Biotechnology) at room temperature for 60 min and washed three times (15 min each) with TBS/ Tween 20. Western blotting luminal reagent (Santa Cruz Biotechnology) was used to visualize peroxidase activity. Controls for nonspeciﬁc binding were determined by omission of the primary antibody. RESULTS Neuronal damage induced by OA was observed using cresyl violet staining. Figure 1A–D showed damaged neuronal morphologic changes in ipsilateral cortex after OA injection. Mechanical neuronal loss and cell debris caused by injection cannula was obvious after OA or vehicle (saline) injection (Fig. 1A and B). The affected areas by OA injection were markedly larger than those by vehicle injection (Fig. 1A and B). In the surrounding of vehicle injection areas, most neurons presented regular morphological characteristics (Fig. 1C). In the surrounding of OA injection areas, however, many neurons displayed cytoplasmic swelling and inﬂammatory cells increased (Fig. 1D). TUNEL staining is a marker for DNA double-strand damage. Double immunoﬂuorescent labeling of TUNEL and AT8 was utilized to determine the relationship of tau hyperphosphorylation and DNA damage caused by OA. It was shown that TUNEL-positive staining existed in affected areas by OA injection and that most TUNEL staining colocalized with AT8 immunostaining (Fig. 1E). Immunohistochemical method was used to detect the dose-response features of AT8, Bcl-2, and Bax immunostaining in affected brain areas by OA injection. Figure 2 illustrated the similar dose-response features of AT8, Bcl-2, and Bax immunostaining induced by OA. At 1 day after serial doses of OA injection, neurons in injection regions displayed AT8, Bcl-2, and Bax immunopositive staining to different extent. Following vehicle or 10 ng OA injection, only a few AT8-, Bcl-2-, and Bax-positive cells existed in injection regions (Fig. 2). In contrast, a lot of AT8-, Bcl-2-, and Bax-positive cells appeared following 20 ng (Fig. 2), 50 ng (data not shown), or 100 ng (Fig. 2) OA injection. Moreover, 100 ng OA injection enlarged the distributional range of AT8-, Bcl-2-, and Bax-positive cells so that the positive cells presented not only in frontal cortex but also in parietal cortex (Fig. 2). From microscopic observations, we also noticed that the distributional range and the number of Bcl-2-positive cells always seemed to be larger or more than that of the AT8- and Bax-positive cells at 1 day following 20 ng (Fig. 2) and 50 ng (data not shown) OA injection (Fig. 2). The immunostaining-positive cells in affected brain areas by OA or vehicle were counted. The results showed that 20, 50, and 100 ng OA injection signiﬁcantly increased the number of AT8-, Bcl-2-, and Bax-positive cells in affected brain areas compared with vehicle and 10 ng OA injection (P ⬍ 0.05, vs. vehicle and 10 ng OA; Fig. 3). Detailed data on the dose-response are listed in the Table 1. Immunohistochemical assay was also used to examine the temporal changes of AT8, Bcl-2, and Bax immuno- Fig. 1. Neuronal damage induced by okadaic acid (OA). A–D: Cresyl violet staining showing damaged neuronal morphological changes after OA injection. The arrows in the low-power photomicrographs (A and B) correspond to those in the high-power photomicrographs (C and D). E: Double staining for TUNEL (ﬂuorescein) and AT8 (rhodamine) showing that DNA damage occurred in neurons with AT8-positive immunostaining after OA injection. 1240 CHEN ET AL. Fig. 2. Photomicrographs showing dose-response features of AT8, Bcl-2, and Bax immunostaining in affected brain areas at 1 day after OA injection. The images were captured from the areas surrounding the injection site. The injection site was immediately on the left edge of each picture. staining in affected brain areas by 20 ng OA injection. Microscopic observations indicated that AT8, Bcl-2, and Bax immunostaining induced by OA (20 ng) had parallel time-courses. In 20 ng OA injection regions, AT8, Bcl-2, and Bax immunostaining all time-dependently changed. The number of the three types of positive cells time-dependently increased at 3 hr, 12 hr, and 1 day, then decreased at 3 days (Fig. 4). AT8, Bcl-2, and Bax staining were all strongest at 1 day and the numbers of all three types of positive cells were largest at 1 day (Fig. 4). In addition, we noted that AT8, Bcl-2, and Bax staining had the intracellular location different from one another. AT8 staining ﬁrst appeared in distal neurites (at 3 hr), then gradually developed toward cell bodies along neuronal processes (at 12 hr), eventually densely accumulated in soma along single axon-like neurites and displayed NFTlike morphology (at 1 day; Fig. 4). Then AT8 staining became weak and the positive neurites shortened or disappeared (at 3 days; Fig. 4). Bax staining was mostly within neuronal soma and proximal neurites (Fig. 4), 1241 INDUCTION OF Bcl-2 AND Bax Fig. 3. Dose-response features of AT8, Bcl-2, and Bax immunostaining induced by OA. Rats were sacriﬁced at 1 day after the doses indicated of OA injection. Values were presented as the sum of positive cells in affected areas per slice and mean ⫾ SEM. Asterisk, signiﬁcant difference from corresponding vehicle and 10 ng OA; number symbol, signiﬁcant difference from corresponding vehicle only (P ⬍ 0.05, one-way ANOVA followed by Student’s t-test). TABLE 1. The number of AT8-, Bcl-2- and Bax-positive cells (cells/slice; means ⴞ S.E.M.) appearing in affected areas by the serial doses of OA injection at 1 d OA (ng) AT8 Bcl-2 Bax 0 10 20 50 100 24 ⫾ 3 21 ⫾ 5 23 ⫾ 6 71 ⫾ 8 162 ⫾ 38 44 ⫾ 12 270 ⫾ 48 484 ⫾ 44 175 ⫾ 29 270 ⫾ 70 553 ⫾ 90 173 ⫾ 46 377 ⫾ 62 435 ⫾ 42 249 ⫾ 44 whereas Bcl-2 staining always appeared within neuronal perinuclear cell bodies (Fig. 4). Cell counting showed the parallel time-courses of AT8, Bcl-2, and Bax immunostaining in affected areas by 20 ng OA injection. Compared with vehicle injection, 20 ng OA injection signiﬁcantly increased the number of AT8-, Bcl-2-, and Baxpositive cells at 12 hr, peaked at 1 day, then declined at 3 and 7 days but remained above the basal level at 3 days (12 hr, 1 day, 3 days; P ⬍ 0.05 vs. vehicle; Fig. 5). Detailed data on the time-course analysis were presented in Table 2. Western blots were applied to analyze the changes in protein levels of Bcl-2 and Bax after OA injection. Figure 6 showed that the protein levels of Bcl-2 and Bax dose- and time-dependently changed. The protein expression of Bcl-2 and Bax increased at 1 day after the serial doses of OA injection (Fig. 6A). The protein levels of Bcl-2 and Bax gradually increased and then decreased after injection of 20 ng OA (Fig. 6B). Using triple immunoﬂuoresent staining combined with confocal laser scanning microscopic analysis, brain slices from rats sacriﬁced at 1 day after 20 ng OA injection were employed to illustrate the topographic relationship between Bcl-2, Bax, and AT8 immunostaining stimulated by OA. In brain areas more adjacent to OA injection site, where neurons showed vulnerability to OA toxicity, AT8 immunostaining was costained with Bcl-2 and Bax immunostaining or only with Bcl-2 immunostaining (Fig. 7). In brain areas less adjacent to OA injection site, where neurons showed resistance to OA toxicity, OA only induced positive staining of Bcl-2 but not that of Bax and AT8 (Fig. 7). DISCUSSION Our results demonstrated that OA could induce neurodegeneration associated with hyperphosphorylation of tau in vivo. Cresyl violet staining showed that OA caused neurodegenreation including neuronal damage and loss. The colocalization of TUNEL staining and AT8 immunostaining illustrated that OA could induce neuronal DNA damage, which was associated with hyperphosphorylation of tau. The observations of neuronal death and hyperphosphorylation of tau induced by OA were consistent with previous studies (Boe et al., 1991; Arendt et al., 1998; Nuydens et al., 1998; Fladmark et al., 1999). The colocalization relationship between neuronal DNA damage and hyperphosphorylation of tau supported a previous report that cytoskeletal pathology including hyperphosphorylation of tau had a major role in OA-induced neuronal death (Tanaka et al., 1998). We examined the effects of OA on protein expression of Bcl-2 and Bax and further compared their features of time-course and dose-response with that of hyperphosphorylation of tau. In the present study, immunohistochemical observations illustrated that quite a few neurons basally expressed low level of Bcl-2 and Bax proteins in rat frontal cortex. The results of both immunohistochemistry and Western blot analysis showed that protein expression of Bcl-2 and Bax was highly induced in rat frontal cortex after OA injection and the induction had features of doseresponse and time-course. Moreover, immunohistochemical results indicated induction of Bcl-2 and Bax expression changed dose- and time-dependently in parallel with hyperphosphorylation of tau stimulated by OA, suggestive of 1242 CHEN ET AL. Fig. 4. Photomicrographs showing temporal changes of AT8, Bcl-2, and Bax immunostaining in affected brain areas after OA (20 ng) injection. INDUCTION OF Bcl-2 AND Bax 1243 Fig. 5. Time-courses of AT8, Bcl-2, and Bax immunostaining induced by OA (20 ng). A–C respectively reﬂect time-courses of AT8, Bcl-2, and Bax immunostainging. Data were expressed as the sum of positive cells in affected areas per slice and represent mean ⫾ SEM. Asterisk, double asterisk, and number symbol: signiﬁcant difference from corresponding vehicle (asterisk, P ⬍ 0.01; double asterisk, P ⬍ 0.001; number symbol, P ⬍ 0.05, Student’s t-test). certain association between hyperphosphorylation of tau and the expression of Bcl-2 and Bax. In respect to doseresponse, these three types of immunostaining all remarkably increased at 1 day after 20, 50, and 100 ng OA injection. As to time-course, all of them markedly in- creased at 12 hr, peaked at 1 day, and reduced at 3 day but remained above the basal level after 20 ng OA injection. These results were signiﬁcant in light of the possibility that integration of apoptotic signals mediated by Bcl-2 and Bax was involved in neurodegenerative process 1244 CHEN ET AL. TABLE 2. The number of AT8-, Bcl-2- and Bax-positive cells (cells/slice; means ⴞ S.E.M.) appearing in affected areas by 20 ng OA injection at 12 h, 1 d and 3 d 12 h AT8 Bcl-2 Bax 1d 3d vehicle OA vehicle OA vehicle OA 19 ⫾ 2 34 ⫾ 8 27 ⫾ 5 102 ⫾ 6 328 ⫾ 20 69 ⫾ 9 24 ⫾ 3 21 ⫾ 5 23 ⫾ 6 270 ⫾ 48 484 ⫾ 44 175 ⫾ 29 30 ⫾ 5 20 ⫾ 5 21 ⫾ 5 92 ⫾ 22 165 ⫾ 16 77 ⫾ 10 Fig. 6. Changes in protein levels of Bcl-2 and Bax after OA injection. Western blots showed that the protein levels of Bcl-2 and Bax altered depending on dose (A) and time-course (B). The protein levels of Bcl-2 and Bax increased at 1 day after the doses indicated of OA injection (A). The protein levels of Bcl-2 and Bax gradually increased and then decreased after injection of 20 ng OA (B). caused by cytoskeletal pathology following PP inhibition with OA. For the ﬁrst time, it was clariﬁed that apoptotic regulatory molecules of Bcl-2 and Bax were highly induced by PP inhibition. But the causes of induction of Bcl-2 and Bax expression by OA were unclear now. Sequentially, the topographic relationship between hyperphosphorylation of tau and inductive expression of Bcl-2 and Bax was determined. Triple immunoﬂuorescent staining delineated that hyperphosphorylation of tau colocalized with the inductive expression of Bcl-2 and Bax or only with the inductive expression of Bcl-2 in areas more adjacent to OA injection site, where neurons were more vulnerable to OA toxicity. However, only Bcl-2 immunostaining was seen, but neither AT8 nor Bax immunostaining in areas less adjacent to OA injection site, where neurons were more resistant to OA toxicity. It has been reported that Bax promotes neuronal death in many neurodegenerative processes and Bcl-2 supports neuronal survival (Krajewski et al., 1995; Martin, 1999; Vila et al., 2001; Raghupathi et al., 2002). Bax only appeared in areas where neurons were more vulnerable to OA toxicity, suggesting that high induction of Bax by OA possibly participated in neuronal death associated with hyperphosphorylation of tau. We noted that there were more cells that express Bcl-2 than those that express Bax. There was substantial Bcl-2 expression even in cells that express Bax. The phenomena were consistent with immunostaining observations that the number of Bcl-2-positive cells was always more than that of Bax-positive cells after injection of the same-dose OA at the same time point. It has been proposed that the ratio of Bcl-2 and Bax is able to decide a cell’s fate under certain condition. Whereas in the areas less adjacent to OA injection site, neurons with few AT8-positive staining showed resistance to OA toxicity and only stained with Bcl-2, but not Bax. It seemed an attempt at cell survival existed after OA treatment. The colocalization of highly expressed Bcl-2 and Bax possibly reﬂected an active antagonism between the two proteins. A previous report showed that Bcl-2 overexpression could Fig. 7. Photographs showing relationship of AT8, Bcl-2, and Bax immunostaining induced by OA (20 ng). The sections are triple-labeled with anti-AT8 (ﬂuorescein), anti-Bcl-2 (rhodamine), and anti-Bax (CY5). INDUCTION OF Bcl-2 AND Bax completely inhibit the death of NGF-differentiated PC12 cells induced by OA by a mechanism that Bcl-2 increased stability of microtubules (Nuydens et al., 2000). But the things may be more complicated. PP has been found to localize at the mitochondrial membrane and to dephosphorylate Bcl-2 (Klumpp et al., 2002). 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