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Catalpol protects primary cultured cortical neurons induced by Aβ1–42 through a mitochondrial-dependent caspase pathway
Neurochemistry International 55 (2009) 741–746
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Catalpol protects primary cultured cortical neurons induced by Ab1–42 through a mitochondrial-dependent caspase pathway Jian Hua Liang a,c, Jing Du a,b, Lian Deng Xu c, Tao Jiang c, Shuang Hao a, Jing Bi a, Bo Jiang a,* a
School of Environmental and Biological Science & Technology, Dalian University of Technology, Dalian, Liaoning, 116024, China Mechanized Infantry College of Shijiazhuang, Shijiazhuang, He Bei, 050083, China c Shenzhen Bao’an District Traditional Chinese Hospital, Shenzhen, Guangdong, 518133, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 July 2009 Accepted 14 July 2009 Available online 22 July 2009
It has been reported that catalpol, an iridoid glucoside, isolated from the root of Rehmannia glutinosa, protected cells from damage induced by a variety of toxic stimulus such as LPS, MPP+ and rotenone. Here, we further evaluated the effect of catalpol against Ab1–42-induced apoptosis in primary cortical neuron cultures. In the present study, the primary cortical neuron culture treated with Ab1–42 was severed as cell model of Alzheimer’s disease (AD) in vitro. By exposure to Ab1–42 (5 mM) for 72 h in cultures, neuronal apoptosis occurred characterized by enhancement of activities of caspases and reactive oxygen species (ROS) as well as Bax increase, loss of mitochondrial membrane potential and cytochrome c release. Pretreatment with catalpol (0.5 mM) for 30 min prior to Ab1–42 treatment attenuated neuronal apoptosis not only by reversing intracellular ROS accumulation, Bax level, mitochondrial membrane potential and, cytochrome c release to some extent, but also through regulating the activity and cleavage of caspase-3 and caspase-9. Thus, catalpol protects primary cultured cortical neurons induced by Ab1–42 through a mitochondrial-dependent caspase pathway. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Catalpol Ab1–42 Caspase Cortical neurons Mitochondria
1. Introduction Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease worldwide in the recent two decades. The clinical symptoms of AD include progressive loss of memory and impairment of cognitive ability (Hauptmann et al., 2006). It is pathologically characterized by extracellular senile plaques, intracellular neurofibrillary tangles and extensive neuronal loss (Selkoe, 1991). In AD, memory loss and cognitive impairment are accompanied by the deterioration of select neuronal population in the hippocampus and cerebral cortex (Morris, 1997). Although difficult to assess the accurate mechanisms due to the nature of the disease, apoptotic neurons have been detected in AD brain and programmed cell death is thought to be one of the key factors contributing to the pathogenesis of AD (Carter, 2008). Apoptosis is a form of cell death characterized by cell shrinkage, plasma membrane blebbing, loss of plasma membrane phospholipid asymmetry, activation of one or more cysteine proteases of the caspase family, mitochondrial membrane depolarization, and mitochondrial oxyradical production, as well as chromatin condensation. b-Amyloid (Ab), accumulation of a 39–43 amino
* Corresponding author. Tel.: +86 411 84706356; fax: +86 411 82139185. E-mail address: [email protected] (B. Jiang). 0197-0186/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2009.07.004
acid peptide, is the primary component of senile plaques and plays a central role in the development of Alzheimer’s disease. In vitro, Ab causes apoptosis in primary neuron cultures (Resende et al., 2007). Ab-induced cell death exhibits typical features of apoptosis: neurite beading, membrane blebbing, chromatin and condensation. Moreover, numerous studies suggested that Ab triggered neuronal cell death via mitochondrial dysfunction (Qiao et al., 2005; Petrosillo et al., 2008), oxidative stress and caspase activation (Allen et al., 2001). The caspases are a family of cysteine proteases and have been widely implicated as essential mediators of many types of neuronal apoptosis, including Ab-mediated cell death. Caspases play an important role in the apoptotic process in two pathways: the extrinsic pathway and the intrinsic pathway. Whichever pathway is involved in, caspase-3 and caspase-9 act as an apoptotic executor in central nervous system disease such as AD (Markus, 2000). Likewise, apoptosis occurs accompanied with mitochondrial dysfunction which results from the imbalance of the excessive production of reactive oxygen species, the loss of mitochondrial membrane potential as well as the release of cytochrome c (Qiao et al., 2005). Nowadays, the aging population is rapidly increasing all over the world. It is essential and necessary to seek for new preventive or therapeutic measures to treat AD. Catalpol, an iridoid glucoside, its chemical structure shown in Fig. 1, isolated from the root of
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J.H. Liang et al. / Neurochemistry International 55 (2009) 741–746 2.4. Immunocytochemical analysis
Fig. 1. The chemical structure of catalpol. Catalpol, isolated from the root of the Chinese traditional herbs Rehmannia glutinosa, is an iridoid glucoside, with a small molecular weight of 362.45.
Rehmannia glutinosa, possesses a broad range of biological and pharmacological activity including purgative, analgesic, sedative, anti-tumor, anti-inflammation and anti-oxidative stress property. The protective effects of catalpol on H2O2, LPS, MPP+ and rotenoneinduced neurotoxicity in vitro (Jiang et al., 2004; Tian et al., 2006, 2007; Bi et al., 2009) and in vivo (Mao et al., 2007; Zhang et al., 2008; Jiang et al., 2008) have been already reported in previous studies. In the present study, it is the first time to apply catalpol in the primary cortical neuron cultures aimed to further investigate its neuroprotective effect against Ab1–42-induced apoptosis and explore the underlying mechanisms of mitochondrial-dependent caspase pathway. 2. Experimental procedures 2.1. Materials Ab1–42 purchased from Chemicon (Recombinant, Escherichia coli), was resuspended in 1% NH4OH and aliquoted at 1 mg/ml as a stock solution stored at 20 8C, according to the manufacture’s instruction. The polyclonal anti-MAP-2(H-300) antibody was purchased from Chemicon. SABC compound kits were from SinoAmerican Biotechnology Company. Mouse antimouse cytochrome C antibody was purchased from PharMingen (San Diego, CA). Rabbit anti-mouse caspase-3, Bax and caspase-9 antibodies were from Cell Signaling Technology (Beverly, MA). Monoclonal anti-b-actin was from Sigma chemical Co. 2.2. Primary cortical neuron cultures and cell treatment Primary cortical neuron cultures were prepared from the cerebral tissues of embryonic day 15/16 mice, according to the method described by Qin et al. (2002). In brief, removed cortex was dissected by mild mechanical trituration and washed with PBS. The dissociated cells were planted onto poly-D-lysine-coated (20 mg/ml) 24-well plates at a density of 5 105/well in Dulbeeeo’s modified Eagle medium/nutrient F12 (DMEM/F12) (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 50 U/ml penicillin, 1.2 g/l sodium bicarbonate and 2mM L-glutamine. The cultures were maintained at 37 8C in a humidified atmosphere of 5% CO2 and 95% air. On 2 days in vitro (DIV2), cultures were treated with 10 mM cytosine b-Darabinofuranoside for additional 48 h in order to suppress the upgrowth of glial cells. Later 48 h, culture medium containing 10 mM cytosine b-D-arabinofuranoside was refreshed. The cultures were >95% pure for cortical neurons identified through immunostaining. On DIV7, the neuronal cultures were used for treatment. Cultures were pretreated with catalpol for 30 min prior to treatment with Ab1–42. 2.3. Cell viability analysis Cell viability was determined using the conventional MTT reduction assay. Briefly, 3-[4,5-dimethylthiazol-2]-2,5 diphenyltetrazolium bromide (MTT) was dissolved in PBS at 5 mg/ml as a stock solution. After Ab1–42 treatment, MTT was added into cultures at a final concentration of 0.5 mg/ml supplemented with fresh DMEM/F12 and incubated with neuronal cells for 4 h. Then, the medium contained MTT was replaced with 200 ml dimethyl sulfoxide (DMSO) per well to dissolve the precipitate. Shaking the 24-well plates for 10 min, DMSO was transferred to a 96well microplate to determine the absorbance at 570 nm in a microplate reader (sunrise, TECAN). Cell viability was expressed as a percentage of the control culture value. At indicated time after cell treatment, the culture supernatant was collected for the subsequent biochemical assays. LDH, as an indicator of cytotoxicity, was released from damaged cells and its leakage was colorimetrically measured by using commercially available kits (Jiancheng Bioengineering), according to the manufacturer’s instruction. Absorbance was read at 440 nm in a spectrophotometer (JASCO, V-560). The release of LDH was calculated as the percentage of control.
Cortical neurons were stained with an antibody against microtubule-associated protein-2 (MAP-2), a marker for the cell body and neurite. Cortical neurons were fixed with 4% paraformaldehyde for 30 min at 37 8C and treated with 3% hydrogen peroxide for another 30 min followed by the incubation with blocking solution for additional 30 min. Afterwards, cultures were incubated with the primary antibody MAP-2 dilution (1:200) for 2 h at 37 8C or overnight at 4 8C. Then, biotinylated secondary antibody was incubated with neurons for 1 h at 37 8C. Subsequently, avidin-biotinylated enzyme complex ABC reagents were added into culture plates for additional 30 min at 37 8C and color was developed with 3,30 -diaminobenzidine (DAB). When color development was appropriate, terminate it with removing DAB and washing the cells with PBS. The images were recorded under 20 objective on an inverted microscope (OLYMPUS IX71) connected to a charge-coupled device camera. The analysis for the quantification of MAP-2-positive neurons and the length of their dendrites was performed through the Image-Pro Plus software installed in the microscope, automatically counting the number and determining the dendrite length in three independent wells of 24-well plates from five to six representative fields per well. 2.5. Detection of caspases activities in cortical neuron cultures Caspase-3 and caspase-9 activities were measured by means of colorimetric Assay kits (Keygen Tech Co., Ltd), according to the manufacturer’s instructions. In brief, cortical neurons were cultured in 35-cm2 T-flasks. After treatment, harvested cells (3–5 106) were incubated with 50 ml lysis buffer (containing 0.5 ml DTT) in an ice bath for 20 min, followed with centrifugating at 10,000 rmp for 1 min at 4 8C. Then, cells were suspended in 50 ml 2 reaction buffer (containing 0.5 ml DTT) and 5 ml caspase-3 or caspase-9 substrate incubating for 4 h at 37 8C. Later, the absorbance was read in a microplate reader (sunrise, TECAN). 2.6. Western blot analysis Cell lysate fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to membranes as described previously. After blocking with 5% horse serum in TBST (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20), membranes were incubated with anti-Bax antibody, anti-cytochrome c antibody, anti-caspase-3, -9 antibody and anti-b-actin antibody followed by horseradish peroxidase-conjugated secondary antibodies. Band were detected using the enhanced chemiluminescence’s light-based detection Kits (Boston, MA, USA). 2.7. Measurement of intracellular reactive oxygen species (ROS) Formation of intracellular ROS was fluorometrically detected using the nonfluorescent probe 20 ,70 -dichlorofluorescein diacetate (DCFH-DA), which diffused into cells where it was oxidized in the presence of ROS into the fluorescent compound 20 ,70 -dichlorofluorescein (DCFH). Then, DCFH reacted with ROS to form the fluorescent product DCF. DCFH-DA was diluted in fresh DMEM/F12 at a final concentration of 10 mM and incubated with neuronal cells for 20 min at 37 8C. Later, loaded cells were washed three times with PBS and the fluorescence intensity of DCF was monitored in a fluorescence plat reader (Genios, TECAN) at 485 nm excitation and 530 nm emission. 2.8. Analysis of mitochondrial membrane potential Changes in mitochondrial membrane potential were estimated by using the fluorescent cationic dye rhodamine 123 (Rh123), which preferentially partitions into active mitochondria based on the highly negative mitochondrial membrane potential. Depolarization of mitochondrial membrane potential results in the loss of Rh123 from the mitochondria and a decrease in intracellular fluorescence. After cell treatment, culture medium in 24-well plates was removed and supplemented with 10 mM Rh123. Rh123 was incubated with cortical neurons for 30 min at 37 8C and then cells were washed with PBS to rinse the unconjuncted dye. The fluorescence was read in a fluorescence plat reader (Genios, TECAN) with excitation length 485 nm and emission length 530 nm. 2.9. Statistical analysis Data are expressed as means S.E.M. with p < 0.05 considered significant. Statistical significance was assayed by one-way analysis of variance (ANOVA) followed by Student’s t-test.
3. Results 3.1. Protective effect of catalpol on cortical neuronal cell viability Primary cortical neuron cultures were exposed to Ab1–42 at a series of concentration (0, 0.5, 1, 2, 5, 10 mM) for 72 h and cell viability was determined by MTT assay. As shown in Fig. 2A, 0.5–
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Image-Pro Plus (IPP) software. Morphological alternations in neuronal cells were shown in Fig. 3A, cultures exposed to Ab1– 42 for 72 h exhibited a 54.43 4.55% decrease in the amount of MAP2-positive neurons and their dendrites averagely shrank to 46.12 5.81% of control (Fig. 3B), showing markedly retractile and tortuous appearances (Fig. 3A) and leading to the neural network formed by dendrites collapse. However, pretreatment with 0.5 mM catalpol for 30 min prior to Ab1–42 not only profoundly attenuated the Ab1–42-induced neuronal loss but also markedly reversed the degeneration of their dendrites. 3.3. Catalpol suppressed pro-apoptotic effects by inhibiting Bax increase, cytochrome c release and caspases cleavage
Fig. 2. (A) Dosage-dependent toxic effect of Ab1–42 on cortical neuronal cell viability. Cortical neuronal cells were exposed to Ab1–42 for 72 h, viable cells were identified by the MTT assay as described in Section 2.3. (B) Effects of catalpol (0.5 mM) against 5 mM Ab1–42-induced cytotoxicity. LDH leakage was determined as an indicator of cytotoxicity and was colorimetrically measured. Data were expressed as means S.E.M. of three independent experiments. *p < 0.05 compared with the cultures treated with 5 mM Ab1–42, ##p < 0.005 compared with control group.
10 mM Ab1–42 induced significant decreases in cell viability in a dose-dependent manner. When Ab1–42 at 5 mM in cultures, neuronal cell viability was 59.42 3.74% in contrast with control samples. Consequently, culture treated with 5 mM Ab1–42 for 72 h was used in the subsequent experimental assays as model group. In previous studies, catalpol at 0.5 mM was approved to be the most effective dosage to protect primary mensencephalic neurons against LPS and MPP+-induced neurotoxicity (Tian et al., 2006, 2007). Therefore, the involvement of catalpol at 0.5 mM in protecting primary cortical neurons from Ab1–42-induced damage was further investigated in our current study. As the apoptotic process, cellular membrane integrity is lost and intracellular LDH inevitably leaks from damaged cells. As shown in Fig. 2B, a mass of LDH release was triggered by Ab1–42. However, when cultures were pretreated with catalpol (0.5 mM), the production of LDH was dramatically reduced. As an indicator of cytotoxicity, LDH correlates with cell viability in some degree. Therefore, from the results presented in Fig. 2, it was concluded that catalpol was effective to protect neurons against the Ab1–42-induced cell damage and greatly improved cell viability. 3.2. Catalpol protects cortical neurons from Ab1–42-induced neurotoxicity Deterioration of cortical neurons was determined by counting the quantification of neuronal loss and measuring their dendrite length following immunocytochemical staining through the
In this study, we demonstrated that changes in the Ab1–42induced Bax were reversed by pretreatment with catalpol at dose of 0.5 mM. Similar results have also been observed in PC12 cells exposed to the H2O2, in which pretreatment with catalpol at 0.1 mM prior to an acute treatment with H2O2 inhibited the upregulation of Bax expression (Jiang et al., 2004). Release of cytochrome c from mitochondria and subsequent activation of caspase-3 represents a key step in the mitochondriondependent apoptotic pathway (Jurgensmeier et al., 1998). To determine whether catalpol exerts its anti-apoptotic action against Ab1–42 via this mechanism, cytochrome c abundance, pro and cleaved caspase-3 and caspase-9 forms were measured. Immunoblotting analysis showed that the cytochrome c expression level in the mitochondrial fraction increased, and decreased in the cytosolic fraction with Ab1–42 treatment, whereas pretreatment with catalpol significantly limited the action. Similarly, Ab1–42 significantly increased caspase-3 cleavage as reflected by increased concentration of cleaved caspase-3, but pretreatment with catalpol could largely inhibit the caspase cleavage. Treatment Ab1–42 resulted in cleavage of the 47-kDa procaspase-9 while pretreatment with catalpol partially counteract the atrophic effect of Ab1–42 (Fig. 4). 3.4. Catalpol blocks the accumulation of ROS and the collapse of mitochondrial membrane potential induced by Ab1–42 in cortical neuron cultures ROS was involved in cell apoptosis through diversified pathways or mechanisms. After treatment with Ab1–42 for 72 h, intracellular ROS generated and the production of ROS was fluorescently measured. As expressed in Fig. 5A, the DCF fluorescence intensity was 157.54 7.78% of control after treatment with 5 mM Ab1–42. However, pretreatment with catalpol caused a corresponding and significant decrease in DCF fluorescence intensity. Changes of mitochondrial membrane potential were estimated using the fluorescent cationic dye Rh123. Results were expressed as a decrease above control basal level, as illustrated in Fig. 5B, mitochondrial membrane potential dropped to 65.32 9.32% of control upon the exposure to Ab1–42, which was expressed by the weakening of the fluorescence intensity of the mitochondrial specific probe Rh123. Pretreatment with catalpol reversed the loss of mitochondrial membrane potential in a significant level (p < 0.05) relative to that of Ab1–42 treatment. 3.5. Catalpol attenuates Ab1–42-induced caspase-3 and caspase-9 activities in cortical neuron cultures In this study, caspase-3 and caspase-9 activities were measured since it is an important biomarker of apoptosis and implicated in the pathogenesis of Ab-induced apoptosis (Allen et al., 2001). The effect of catalpol on lowering caspase-3 activity by exposure to
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Fig. 3. (A) Protective effect of catalpol on Ab1–42-induced neurotoxicity in cortical neuronal cells. Immunocytochemical analysis of the effect of catalpol on Ab1–42-induced degeneration to cortical neurons. After treatment, neurons were immunostained with anti-MAP-2 antibody. The amount of MAP-2-positive neurons was less and their dendrites were tortuous and retractile in cultures treated with Ab1–42. However, catalpol dramatically ameliorated the detrimental effect triggered by Ab1–42. (B) Degeneration of cortical neurons was estimated by counting MAP-2-positive neurons and measuring the length of dendrites after treatment. Data are expressed as means S.E.M. of three independent experiments. *p < 0.05 compared with cultures exposed to 5 mM Ab1–42, #p < 0.05 compared with control. Scar bar: 100 mm.
Ab1–42 was presented in Fig. 4A. After treatment, neuronal cells underwent apoptosis and caspase-3 activity was up-regulated to 250.00 27.95% compared with control group. While, the caspase-3 activity was lowered to 152.10 24.16% relative to control after the pretreatment with catalpol, which was indicative that neuronal apoptosis was partially inhibited. Caspase-9 also was inhibited by catalpol after Ab1–42-induced increasing. Caspase-9 activity was upregulated to 253.22 17.21% compared with control group. While, the caspase-9 activity was lowered to 150.10 11.36% relative to control after the pretreatment with catalpol (Fig. 6B).
Fig. 4. Western blot analysis of apoptotic protein. Showing Bax (21 kDa), cytochrome c (15 kDa) release into cytosolic fraction from the mitochondria, cleavage of pro-caspase-3 (32 kDa) into the active (20 kDa) form; cleavage of procaspase-9 (47 kDa) into the active forms. Reprobing for b-actin served as internal loading control. Western blot data are representative of three independent experiments with similar results.
4. Discussion In the present study, we found that exposure of cortical neurons to catalpol attenuated Ab1–42-induced apoptosis mainly via mitochondrial-dependent caspase cascade. The effects were visualized by using a wide range of different parameters including MTT cell viability and LDH cytotoxicity assay, ROS accumulation assay, mitochondrial membrane potential and cytochrome c determination, caspase-3 and caspase-9 analysis as well as morphological analysis in neuronal cells. Since Ab-induced apoptosis results in neurodegeneration, our results suggested that catalpol, isolated from the Chinese traditional herb R. glutinosa, might be potentially an anti-apoptotic agent against neurodegeneration. It is generally recognized that application of Ab to cultured cortical or hippocampal neurons mimics the pathogenesis of AD and Ab contributes to neurodegeneration in AD through activation of an apoptotic pathway (Carter, 2008). In our present study, the cortical neuron culture treated with 5 mM Ab1–42 for 72 h was served as AD cell model and the neuroprotective effect of catalpol on AD was investigated. First of all, it is well known that the progressive deterioration of hippocampal and cortical neurons emerges in brains of AD. Exposure to Ab1–42 resulted in an obvious damage in cortical neurons, which was visualized after immunocytochemical staining. Pretreatment with catalpol rescued cortical neurons from Ab1–42-induced neurotoxicity, which was revealed by morphological observation after immunocytochemical staining and also further confirmed by LDH cytotoxicity assay. The Bcl-2 family of proteins, an important endogenous regulator in cellular activity after a variety of physiological and pathological insults, has been suggested to be directly dependent on the elevation of Bax and its translocation to the mitochondrial
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Fig. 5. Effects of catalpol on the elevation of the accumulation of intracellular ROS, and the loss of mitochondria membrane potential due to Ab1–42 treatment. After treatment with 5 mM Ab1–42 in the presence or absence of 0.5 mM catalpol for 72 h, the three parameters were wholly assayed by fluorescent measurement. The changes of the three parameters were expressed as the percentage of control. (A). The production of intracellular ROS increased compared with control after Ab1–42 treatment and the administration of catalpol resulted in a dramatic decrease. (B). Catalpol significantly rescued mitochondrial membrane potential from dropping. Data were expressed as means S.E.M. of three independent experiments. *p < 0.05 compared with the 5 mM Ab1–42-treated cultures, ##p < 0.01 compared with control samples.
membrane (Kazmierczak et al., 2008). After translocated to the mitochondrial membrane, Bax can homodimerize and activate the terminal caspases by alteration of mitochondrial functions, resulting in the release of apoptosis promoting factors into the cytoplasm (Yuan and Yankner, 2000). Obviously, the protective effects of catalpol against Ab1–42 toxicity observed here are, at least in part, attributed to its ability to suppress up-regulation of the pro-apoptotic protein Bax. Our previous studies were consist with this result that Bax expression is reduced by catalpol in PC12 cells with an acute H2O2 treatment and catalpol modulates the expressions of Bax and attenuates apoptosis in gerbils after ischemic injury(Jiang et al., 2004; Li et al., 2006). Mitochondrial dysfunction, the excessive production of reactive oxygen species, and the decrease of mitochondrial membrane potential have long been implicated in the process of apoptosis (Zhang et al., 2007). It is generally accepted that the opening of mitochondrial permeability transition pore (MPTP) contributes to mitochondrial dysfunction, resulting in osmotic swelling of mitochondrial matrix, dissipation of the membrane potential, cessation of the ATP synthesis, the release of cytochrome c (Agostinho et al., 2008). ROS such as hydrogen peroxide, superoxide anion and hydroxyl radical readily damages biological molecules, ultimately results in apoptotic or necrotic cell death
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Fig. 6. Caspase-3 and caspase-9 activities in cortical neurons. Cultures were pretreated with catalpol (0.5 mM) for 30 min prior to 5 mM Ab1–42 for 72 h. After treatment, caspase-3, -9 activities were measured. The values were expressed as a percentage of caspase-3 or caspase-9 activities of control cultures and are the means S.E.M. of three independent experiments. *p < 0.05, **p < 0.01 compared with cultures exposed to 5 mM Ab1–42, ##p < 0.005 compared with control.
and is involved in the apoptotic mechanism of Ab-mediated neurotoxicity (Behl et al., 1994). The important role of mitochondria in apoptotic cell death has been reinforced by study showing the contribution of ROS in cell death signaling (Fleury et al., 2002). The overproduction of ROS, mainly produced by mitochondria, leads to oxidative stress and cellular oxidative pathways that proceed through apoptosis appear to be a predominant factor in the cell loss observed during Alzheimer’s disease (Chong et al., 2005). In the current study, Ab1–42 triggered significant accumulation of intracellular ROS as well as the overproduction of ROS further led to mitochondrial dysfunction, which ultimately resulted in apoptosis. Rising evidence confirmed that ROS is extensive in the AD brains, and plays a key role in Ab-induced neuronal cell death (Miranda et al., 2000). In agreement with the research, this study demonstrated that Ab1–42 induces ROS generation, and the intracellular ROS was scavenged by catalpol at 0.5 mM. As Somayajulu et al. (2005) proposed, mitochondrial control of apoptosis occurs at two levels, the maintenance of ATP production and mitochondrial membrane potential. The collapse of mitochondrial membrane potential was occurred in mitochondria level after Ab1–42-induced apoptosis, in agreement with previous study (Wang et al., 2001). Disruption of mitochondrial membrane potential by reactive oxygen species may release apoptosis-inducing factors which activate caspase cascade, cause nuclear condensation, and generate secondary reactive oxygen species (Petit et al., 1996). The release of cytochrome c from
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mitochondria to the cytosol is essential for caspase activation and cleavage. In the present study, we have shown that catalpol inhibited Ab1–42-induced cytochrome c release and the cleavage of caspase-3 and caspase-9. The activation of caspase-3 is also believed to be important for commitment to or execution of neuronal apoptosis (Harada and Sugimoto, 1999). The suppressive effect of catalpol on caspase-3 and caspase-9 activity further suggests that the protective effect of catalpol on cell death is related to its mitochondrial-dependent caspase pathway. In summary, catalpol attenuated neuronal apoptosis not only by reversing intracellular ROS accumulation, Bax level, mitochondrial dysfunction to some extent, but also through regulating the expression of caspase-3 and caspase-9. Thus, catalpol protects primary cultured cortical neurons induced by Ab1–42 through a mitochondrial-dependent caspase pathway. Taken together, catalpol effectively protects cortical neuronal cells from the Ab1–42induced neurotoxicity and suggest a neuroprotective effect on Ab1–42 insult. Acknowledgements This work was supported by grants from the Natural Science Foundation of Liaoning (20072185) and Science and Technology Project of Dalian (2008E11SF163). References Agostinho, P., Lopes, J.P., Velez, Z., Oliveira, C.R., 2008. Overactivation of calcineurin induced by amyloid-beta and prion proteins. Neurochem. Int. 52 (6), 1226– 1233. Allen, J.W., Eeldadah, B.A., Huang, X.L., Knoblach, S.M., Faden, A.I., 2001. Multiple caspases are involved in b-amyloid-induced neuronal apoptosis. J. Neurosci. Res. 65, 45–53. Behl, C., Davis, J.B., Lesley, R., Schubert, D., 1994. Hydrogen peroxide mediates amyloid bprotein toxicity. Cell 77, 817–827. Bi, J., Jiang, B., Hao, S., Zhang, A., Dong, Y., Jiang, T., An, L.J., 2009. Catalpol attenuates nitric oxide increase via ERK signaling pathways induced by rotenone in mesencephalic neurons. Neurochem. Int. 54, 264–270. Carter, C.J., 2008. Interactions between the products of the Herpes simplex genome and Alzheimer’s disease susceptibility genes: relevance to pathological-signalling cascades. Neurochem. Int. 52 (6), 920–934. Chong, Z.Z., Li, F., Maiese, K., 2005. Stress in the brain: novel cellular mechanisms of injury liked to Alzheimer’s disease. Brain Res. Rev. 49, 1–21. Fleury, C., Mignotte, B., Vayssiere, J.L., 2002. Mitochondrial reactive oxygen species in cell death signaling. Biochimie 84, 131–141. Harada, J., Sugimoto, M., 1999. Activation of caspase-3 in beta-amyloid-induced apoptosis of cultured rat cortical neurons. Brain Res. 842, 311–323. Hauptmann, S., Keil, U., Scherping, I., Bonert, A., Eckert, A., Muller, W.E., 2006. Mitochondrial dysfunction in sporadic and genetic Alzheimer’s disease. Exp. Gerontol. 41, 668–673. Jiang, B., Liu, J.H., Bao, Y.M., An, L.J., 2004. Catalpol inhibits apoptosis in hydrogen peroxide-induced PC12 cells by preventing cytochrome c release and inactivating of caspase cascade. Toxicon 43, 53–59.
Jiang, B., Zhang, H., Bing, J., Zhang, X.L., 2008. Neuroprotective activities of catalpol on MPP+/MPTP-induced neurotoxicity. Neurol. Res. 30, 639–644. Jurgensmeier, J.M., Xie, Z., Deveraux, Q., Ellerby, L., Bredesen, D., Reed, J.C., 1998. Bax directly induces release of cytochrome c from isolated mitochondria. Proc. Natl. Acad. Sci. U.S.A 95, 4997–5002. Kazmierczak, A., Strosznajder, J.B., Adamczyk, A., 2008. alpha-Synuclein enhances secretion and toxicity of amyloid beta peptides in PC12 cells. Neurochem. Int. 53 (6–8), 263–269. Li, D.Q., Bao, Y.M., Li, Y., Wang, C.F., Liu, Y.X., An, L.J., 2006. Catalpol modulates the expressions of Bcl-2 and Bax and attenuates apoptosis in gerbils after ischemic injury. Brain Res. 1115, 179–185. Mao, Y.R., Jiang, L., Duan, Y.L., Jiang, B., An, L.J., 2007. Efficacy of catalpol as protectant against oxidative stress and mitochondrial dysfunction on rotenone-induced toxicity in mice brain. Toxicol. Pharmacol. 23, 314–318. ˜ oz, F.J., Ruiz, F., Leighton, F., Inestrosa, N.C., Miranda, S., Opazo, C., Larrondo, L.F., Mun 2000. The role of oxidative stress in the toxicity induced by amyloid b-peptide in Alzheimer’s disease. Prog. Neurobiol. 62, 633–648. Markus, G.G., 2000. Caspase: key players in programmed cell death. Curr. Opin. Struct. Biol. 10, 649–655. Morris, J.C., 1997. The challenge of characterizing normal brain aging in relation to Alzheimer’s disease. Neurobiol. Aging 18 (4), 388–389. Petit, P.X., Susin, S.A., Zamzami, N., Mignotte, B., Kroemer, G., 1996. Mitochondria and programmed cell death: back to the future. FEBS Lett. 396, 7–13. Petrosillo, G., Matera, M., Casanova, G., Ruggiero, F.M., Paradies, G., 2008. Mitochondrial dysfunction in rat brain with aging involvement of complex I, reactive oxygen species and cardiolipin. Neurochem. Int. 53 (5), 126–131. Qiao, H.J., Koya, R.C., Nakagawa, K., Tanaka, H., Fujita, H., Takitmoto, M., Kuzamaki, N., 2005. Inhibition of Alzheimer’s amyloid-peptide-induced reduction of mitochondrial membrane potential and neurotoxicity by gelsolin. Neurobiol. Aging 26, 849–855. Qin, L.Y., Liu, Y.X., Cooper, C.L., Liu, B., Wilso, B., Hong, J.S., 2002. The role of microglia in beta-amyloid(1–42) toxicity to cortical and mesencephalic neurons. J. Neurochem. 83, 973–983. Resende, R., Pereira, C., Agostinho, P., Vieira, A.P., Malva, J.O., Oliveira, C.R., 2007. Susceptibility of hippocampal neurons to Ab peptide toxicity is associated with perturbation of Ca2+ homeostasis. Brain Res. 1143, 11–21. Selkoe, D.J., 1991. The molecular pathology of Alzheimer’s disease. Neuron 6, 487– 498. Somayajulu, M., McCarthy, S., Hung, M., Sikorska, M., Borowy-Borowski, H., Pandey, S., 2005. Role of mitochondria in neuronal cell death induced by oxidative stress: neuroprotection by coenzyme Q10. Neurobiol. Dis. 18, 618– 627. Tian, Y.Y., An, L.J., Jiang, L., Duan, Y.L., Chen, J., Jiang, B., 2006. Catalpol protects dopaminergic neurons from LPS-induced neurotoxicity in mesencephalic neuron-glia cultures. Life Sci. 80 (3), 193–199. Tian, Y.Y., Jiang, B., An, L.J., Bao, Y.M., 2007. Neuroprotective effect of catalpol against MPP+-induced oxidative stress in mesencephalic neurons. Eur. J. Pharmacol. 568 (1–3), 142–148. Wang, C.N., Chi, C.W., Lin, Y.L., Chen, C.F., Shiao, Y.J., 2001. The neuroprotective effects of phytoestrogens on amyloid b protein-induced toxicity are mediated by abrogating the activation of caspase cascade in rat cortical neurons. J. Biol. Chem. 276, 5287–5295. Yuan, J.Y., Yankner, B.A., 2000. Apoptosis in the nervous system. Nature 47 (10), 802–809. Zhang, L., Yu, H.X., Sun, Y., Lin, X.F., Chen, B., Tan, C., Cao, G.X., Wang, Z.W., 2007. Protective effects of salidroside on hydrogen peroxide-induced apoptosis in SH-SYSY human neuroblastoma cells. Eur. J. Pharmacol. 564 (1–3), 18–25. Zhang, X.L., An, L.J., Bao, Y.M., Wang, J.Y., Jiang, B., 2008. D-Galactose administration induces memory loss and energy metabolism disturbance in mice: protective effects of catalpol. Food Chem. Toxicol. 462, 888–894.
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Catalpol protects primary cultured cortical neurons induced by Ab1–42 through a mitochondrial-dependent caspase pathway Jian Hua Liang a,c, Jing Du a,b, Lian Deng Xu c, Tao Jiang c, Shuang Hao a, Jing Bi a, Bo Jiang a,* a
School of Environmental and Biological Science & Technology, Dalian University of Technology, Dalian, Liaoning, 116024, China Mechanized Infantry College of Shijiazhuang, Shijiazhuang, He Bei, 050083, China c Shenzhen Bao’an District Traditional Chinese Hospital, Shenzhen, Guangdong, 518133, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 July 2009 Accepted 14 July 2009 Available online 22 July 2009
It has been reported that catalpol, an iridoid glucoside, isolated from the root of Rehmannia glutinosa, protected cells from damage induced by a variety of toxic stimulus such as LPS, MPP+ and rotenone. Here, we further evaluated the effect of catalpol against Ab1–42-induced apoptosis in primary cortical neuron cultures. In the present study, the primary cortical neuron culture treated with Ab1–42 was severed as cell model of Alzheimer’s disease (AD) in vitro. By exposure to Ab1–42 (5 mM) for 72 h in cultures, neuronal apoptosis occurred characterized by enhancement of activities of caspases and reactive oxygen species (ROS) as well as Bax increase, loss of mitochondrial membrane potential and cytochrome c release. Pretreatment with catalpol (0.5 mM) for 30 min prior to Ab1–42 treatment attenuated neuronal apoptosis not only by reversing intracellular ROS accumulation, Bax level, mitochondrial membrane potential and, cytochrome c release to some extent, but also through regulating the activity and cleavage of caspase-3 and caspase-9. Thus, catalpol protects primary cultured cortical neurons induced by Ab1–42 through a mitochondrial-dependent caspase pathway. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Catalpol Ab1–42 Caspase Cortical neurons Mitochondria
1. Introduction Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease worldwide in the recent two decades. The clinical symptoms of AD include progressive loss of memory and impairment of cognitive ability (Hauptmann et al., 2006). It is pathologically characterized by extracellular senile plaques, intracellular neurofibrillary tangles and extensive neuronal loss (Selkoe, 1991). In AD, memory loss and cognitive impairment are accompanied by the deterioration of select neuronal population in the hippocampus and cerebral cortex (Morris, 1997). Although difficult to assess the accurate mechanisms due to the nature of the disease, apoptotic neurons have been detected in AD brain and programmed cell death is thought to be one of the key factors contributing to the pathogenesis of AD (Carter, 2008). Apoptosis is a form of cell death characterized by cell shrinkage, plasma membrane blebbing, loss of plasma membrane phospholipid asymmetry, activation of one or more cysteine proteases of the caspase family, mitochondrial membrane depolarization, and mitochondrial oxyradical production, as well as chromatin condensation. b-Amyloid (Ab), accumulation of a 39–43 amino
* Corresponding author. Tel.: +86 411 84706356; fax: +86 411 82139185. E-mail address: [email protected] (B. Jiang). 0197-0186/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2009.07.004
acid peptide, is the primary component of senile plaques and plays a central role in the development of Alzheimer’s disease. In vitro, Ab causes apoptosis in primary neuron cultures (Resende et al., 2007). Ab-induced cell death exhibits typical features of apoptosis: neurite beading, membrane blebbing, chromatin and condensation. Moreover, numerous studies suggested that Ab triggered neuronal cell death via mitochondrial dysfunction (Qiao et al., 2005; Petrosillo et al., 2008), oxidative stress and caspase activation (Allen et al., 2001). The caspases are a family of cysteine proteases and have been widely implicated as essential mediators of many types of neuronal apoptosis, including Ab-mediated cell death. Caspases play an important role in the apoptotic process in two pathways: the extrinsic pathway and the intrinsic pathway. Whichever pathway is involved in, caspase-3 and caspase-9 act as an apoptotic executor in central nervous system disease such as AD (Markus, 2000). Likewise, apoptosis occurs accompanied with mitochondrial dysfunction which results from the imbalance of the excessive production of reactive oxygen species, the loss of mitochondrial membrane potential as well as the release of cytochrome c (Qiao et al., 2005). Nowadays, the aging population is rapidly increasing all over the world. It is essential and necessary to seek for new preventive or therapeutic measures to treat AD. Catalpol, an iridoid glucoside, its chemical structure shown in Fig. 1, isolated from the root of
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Fig. 1. The chemical structure of catalpol. Catalpol, isolated from the root of the Chinese traditional herbs Rehmannia glutinosa, is an iridoid glucoside, with a small molecular weight of 362.45.
Rehmannia glutinosa, possesses a broad range of biological and pharmacological activity including purgative, analgesic, sedative, anti-tumor, anti-inflammation and anti-oxidative stress property. The protective effects of catalpol on H2O2, LPS, MPP+ and rotenoneinduced neurotoxicity in vitro (Jiang et al., 2004; Tian et al., 2006, 2007; Bi et al., 2009) and in vivo (Mao et al., 2007; Zhang et al., 2008; Jiang et al., 2008) have been already reported in previous studies. In the present study, it is the first time to apply catalpol in the primary cortical neuron cultures aimed to further investigate its neuroprotective effect against Ab1–42-induced apoptosis and explore the underlying mechanisms of mitochondrial-dependent caspase pathway. 2. Experimental procedures 2.1. Materials Ab1–42 purchased from Chemicon (Recombinant, Escherichia coli), was resuspended in 1% NH4OH and aliquoted at 1 mg/ml as a stock solution stored at 20 8C, according to the manufacture’s instruction. The polyclonal anti-MAP-2(H-300) antibody was purchased from Chemicon. SABC compound kits were from SinoAmerican Biotechnology Company. Mouse antimouse cytochrome C antibody was purchased from PharMingen (San Diego, CA). Rabbit anti-mouse caspase-3, Bax and caspase-9 antibodies were from Cell Signaling Technology (Beverly, MA). Monoclonal anti-b-actin was from Sigma chemical Co. 2.2. Primary cortical neuron cultures and cell treatment Primary cortical neuron cultures were prepared from the cerebral tissues of embryonic day 15/16 mice, according to the method described by Qin et al. (2002). In brief, removed cortex was dissected by mild mechanical trituration and washed with PBS. The dissociated cells were planted onto poly-D-lysine-coated (20 mg/ml) 24-well plates at a density of 5 105/well in Dulbeeeo’s modified Eagle medium/nutrient F12 (DMEM/F12) (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 50 U/ml penicillin, 1.2 g/l sodium bicarbonate and 2mM L-glutamine. The cultures were maintained at 37 8C in a humidified atmosphere of 5% CO2 and 95% air. On 2 days in vitro (DIV2), cultures were treated with 10 mM cytosine b-Darabinofuranoside for additional 48 h in order to suppress the upgrowth of glial cells. Later 48 h, culture medium containing 10 mM cytosine b-D-arabinofuranoside was refreshed. The cultures were >95% pure for cortical neurons identified through immunostaining. On DIV7, the neuronal cultures were used for treatment. Cultures were pretreated with catalpol for 30 min prior to treatment with Ab1–42. 2.3. Cell viability analysis Cell viability was determined using the conventional MTT reduction assay. Briefly, 3-[4,5-dimethylthiazol-2]-2,5 diphenyltetrazolium bromide (MTT) was dissolved in PBS at 5 mg/ml as a stock solution. After Ab1–42 treatment, MTT was added into cultures at a final concentration of 0.5 mg/ml supplemented with fresh DMEM/F12 and incubated with neuronal cells for 4 h. Then, the medium contained MTT was replaced with 200 ml dimethyl sulfoxide (DMSO) per well to dissolve the precipitate. Shaking the 24-well plates for 10 min, DMSO was transferred to a 96well microplate to determine the absorbance at 570 nm in a microplate reader (sunrise, TECAN). Cell viability was expressed as a percentage of the control culture value. At indicated time after cell treatment, the culture supernatant was collected for the subsequent biochemical assays. LDH, as an indicator of cytotoxicity, was released from damaged cells and its leakage was colorimetrically measured by using commercially available kits (Jiancheng Bioengineering), according to the manufacturer’s instruction. Absorbance was read at 440 nm in a spectrophotometer (JASCO, V-560). The release of LDH was calculated as the percentage of control.
Cortical neurons were stained with an antibody against microtubule-associated protein-2 (MAP-2), a marker for the cell body and neurite. Cortical neurons were fixed with 4% paraformaldehyde for 30 min at 37 8C and treated with 3% hydrogen peroxide for another 30 min followed by the incubation with blocking solution for additional 30 min. Afterwards, cultures were incubated with the primary antibody MAP-2 dilution (1:200) for 2 h at 37 8C or overnight at 4 8C. Then, biotinylated secondary antibody was incubated with neurons for 1 h at 37 8C. Subsequently, avidin-biotinylated enzyme complex ABC reagents were added into culture plates for additional 30 min at 37 8C and color was developed with 3,30 -diaminobenzidine (DAB). When color development was appropriate, terminate it with removing DAB and washing the cells with PBS. The images were recorded under 20 objective on an inverted microscope (OLYMPUS IX71) connected to a charge-coupled device camera. The analysis for the quantification of MAP-2-positive neurons and the length of their dendrites was performed through the Image-Pro Plus software installed in the microscope, automatically counting the number and determining the dendrite length in three independent wells of 24-well plates from five to six representative fields per well. 2.5. Detection of caspases activities in cortical neuron cultures Caspase-3 and caspase-9 activities were measured by means of colorimetric Assay kits (Keygen Tech Co., Ltd), according to the manufacturer’s instructions. In brief, cortical neurons were cultured in 35-cm2 T-flasks. After treatment, harvested cells (3–5 106) were incubated with 50 ml lysis buffer (containing 0.5 ml DTT) in an ice bath for 20 min, followed with centrifugating at 10,000 rmp for 1 min at 4 8C. Then, cells were suspended in 50 ml 2 reaction buffer (containing 0.5 ml DTT) and 5 ml caspase-3 or caspase-9 substrate incubating for 4 h at 37 8C. Later, the absorbance was read in a microplate reader (sunrise, TECAN). 2.6. Western blot analysis Cell lysate fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to membranes as described previously. After blocking with 5% horse serum in TBST (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20), membranes were incubated with anti-Bax antibody, anti-cytochrome c antibody, anti-caspase-3, -9 antibody and anti-b-actin antibody followed by horseradish peroxidase-conjugated secondary antibodies. Band were detected using the enhanced chemiluminescence’s light-based detection Kits (Boston, MA, USA). 2.7. Measurement of intracellular reactive oxygen species (ROS) Formation of intracellular ROS was fluorometrically detected using the nonfluorescent probe 20 ,70 -dichlorofluorescein diacetate (DCFH-DA), which diffused into cells where it was oxidized in the presence of ROS into the fluorescent compound 20 ,70 -dichlorofluorescein (DCFH). Then, DCFH reacted with ROS to form the fluorescent product DCF. DCFH-DA was diluted in fresh DMEM/F12 at a final concentration of 10 mM and incubated with neuronal cells for 20 min at 37 8C. Later, loaded cells were washed three times with PBS and the fluorescence intensity of DCF was monitored in a fluorescence plat reader (Genios, TECAN) at 485 nm excitation and 530 nm emission. 2.8. Analysis of mitochondrial membrane potential Changes in mitochondrial membrane potential were estimated by using the fluorescent cationic dye rhodamine 123 (Rh123), which preferentially partitions into active mitochondria based on the highly negative mitochondrial membrane potential. Depolarization of mitochondrial membrane potential results in the loss of Rh123 from the mitochondria and a decrease in intracellular fluorescence. After cell treatment, culture medium in 24-well plates was removed and supplemented with 10 mM Rh123. Rh123 was incubated with cortical neurons for 30 min at 37 8C and then cells were washed with PBS to rinse the unconjuncted dye. The fluorescence was read in a fluorescence plat reader (Genios, TECAN) with excitation length 485 nm and emission length 530 nm. 2.9. Statistical analysis Data are expressed as means S.E.M. with p < 0.05 considered significant. Statistical significance was assayed by one-way analysis of variance (ANOVA) followed by Student’s t-test.
3. Results 3.1. Protective effect of catalpol on cortical neuronal cell viability Primary cortical neuron cultures were exposed to Ab1–42 at a series of concentration (0, 0.5, 1, 2, 5, 10 mM) for 72 h and cell viability was determined by MTT assay. As shown in Fig. 2A, 0.5–
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Image-Pro Plus (IPP) software. Morphological alternations in neuronal cells were shown in Fig. 3A, cultures exposed to Ab1– 42 for 72 h exhibited a 54.43 4.55% decrease in the amount of MAP2-positive neurons and their dendrites averagely shrank to 46.12 5.81% of control (Fig. 3B), showing markedly retractile and tortuous appearances (Fig. 3A) and leading to the neural network formed by dendrites collapse. However, pretreatment with 0.5 mM catalpol for 30 min prior to Ab1–42 not only profoundly attenuated the Ab1–42-induced neuronal loss but also markedly reversed the degeneration of their dendrites. 3.3. Catalpol suppressed pro-apoptotic effects by inhibiting Bax increase, cytochrome c release and caspases cleavage
Fig. 2. (A) Dosage-dependent toxic effect of Ab1–42 on cortical neuronal cell viability. Cortical neuronal cells were exposed to Ab1–42 for 72 h, viable cells were identified by the MTT assay as described in Section 2.3. (B) Effects of catalpol (0.5 mM) against 5 mM Ab1–42-induced cytotoxicity. LDH leakage was determined as an indicator of cytotoxicity and was colorimetrically measured. Data were expressed as means S.E.M. of three independent experiments. *p < 0.05 compared with the cultures treated with 5 mM Ab1–42, ##p < 0.005 compared with control group.
10 mM Ab1–42 induced significant decreases in cell viability in a dose-dependent manner. When Ab1–42 at 5 mM in cultures, neuronal cell viability was 59.42 3.74% in contrast with control samples. Consequently, culture treated with 5 mM Ab1–42 for 72 h was used in the subsequent experimental assays as model group. In previous studies, catalpol at 0.5 mM was approved to be the most effective dosage to protect primary mensencephalic neurons against LPS and MPP+-induced neurotoxicity (Tian et al., 2006, 2007). Therefore, the involvement of catalpol at 0.5 mM in protecting primary cortical neurons from Ab1–42-induced damage was further investigated in our current study. As the apoptotic process, cellular membrane integrity is lost and intracellular LDH inevitably leaks from damaged cells. As shown in Fig. 2B, a mass of LDH release was triggered by Ab1–42. However, when cultures were pretreated with catalpol (0.5 mM), the production of LDH was dramatically reduced. As an indicator of cytotoxicity, LDH correlates with cell viability in some degree. Therefore, from the results presented in Fig. 2, it was concluded that catalpol was effective to protect neurons against the Ab1–42-induced cell damage and greatly improved cell viability. 3.2. Catalpol protects cortical neurons from Ab1–42-induced neurotoxicity Deterioration of cortical neurons was determined by counting the quantification of neuronal loss and measuring their dendrite length following immunocytochemical staining through the
In this study, we demonstrated that changes in the Ab1–42induced Bax were reversed by pretreatment with catalpol at dose of 0.5 mM. Similar results have also been observed in PC12 cells exposed to the H2O2, in which pretreatment with catalpol at 0.1 mM prior to an acute treatment with H2O2 inhibited the upregulation of Bax expression (Jiang et al., 2004). Release of cytochrome c from mitochondria and subsequent activation of caspase-3 represents a key step in the mitochondriondependent apoptotic pathway (Jurgensmeier et al., 1998). To determine whether catalpol exerts its anti-apoptotic action against Ab1–42 via this mechanism, cytochrome c abundance, pro and cleaved caspase-3 and caspase-9 forms were measured. Immunoblotting analysis showed that the cytochrome c expression level in the mitochondrial fraction increased, and decreased in the cytosolic fraction with Ab1–42 treatment, whereas pretreatment with catalpol significantly limited the action. Similarly, Ab1–42 significantly increased caspase-3 cleavage as reflected by increased concentration of cleaved caspase-3, but pretreatment with catalpol could largely inhibit the caspase cleavage. Treatment Ab1–42 resulted in cleavage of the 47-kDa procaspase-9 while pretreatment with catalpol partially counteract the atrophic effect of Ab1–42 (Fig. 4). 3.4. Catalpol blocks the accumulation of ROS and the collapse of mitochondrial membrane potential induced by Ab1–42 in cortical neuron cultures ROS was involved in cell apoptosis through diversified pathways or mechanisms. After treatment with Ab1–42 for 72 h, intracellular ROS generated and the production of ROS was fluorescently measured. As expressed in Fig. 5A, the DCF fluorescence intensity was 157.54 7.78% of control after treatment with 5 mM Ab1–42. However, pretreatment with catalpol caused a corresponding and significant decrease in DCF fluorescence intensity. Changes of mitochondrial membrane potential were estimated using the fluorescent cationic dye Rh123. Results were expressed as a decrease above control basal level, as illustrated in Fig. 5B, mitochondrial membrane potential dropped to 65.32 9.32% of control upon the exposure to Ab1–42, which was expressed by the weakening of the fluorescence intensity of the mitochondrial specific probe Rh123. Pretreatment with catalpol reversed the loss of mitochondrial membrane potential in a significant level (p < 0.05) relative to that of Ab1–42 treatment. 3.5. Catalpol attenuates Ab1–42-induced caspase-3 and caspase-9 activities in cortical neuron cultures In this study, caspase-3 and caspase-9 activities were measured since it is an important biomarker of apoptosis and implicated in the pathogenesis of Ab-induced apoptosis (Allen et al., 2001). The effect of catalpol on lowering caspase-3 activity by exposure to
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Fig. 3. (A) Protective effect of catalpol on Ab1–42-induced neurotoxicity in cortical neuronal cells. Immunocytochemical analysis of the effect of catalpol on Ab1–42-induced degeneration to cortical neurons. After treatment, neurons were immunostained with anti-MAP-2 antibody. The amount of MAP-2-positive neurons was less and their dendrites were tortuous and retractile in cultures treated with Ab1–42. However, catalpol dramatically ameliorated the detrimental effect triggered by Ab1–42. (B) Degeneration of cortical neurons was estimated by counting MAP-2-positive neurons and measuring the length of dendrites after treatment. Data are expressed as means S.E.M. of three independent experiments. *p < 0.05 compared with cultures exposed to 5 mM Ab1–42, #p < 0.05 compared with control. Scar bar: 100 mm.
Ab1–42 was presented in Fig. 4A. After treatment, neuronal cells underwent apoptosis and caspase-3 activity was up-regulated to 250.00 27.95% compared with control group. While, the caspase-3 activity was lowered to 152.10 24.16% relative to control after the pretreatment with catalpol, which was indicative that neuronal apoptosis was partially inhibited. Caspase-9 also was inhibited by catalpol after Ab1–42-induced increasing. Caspase-9 activity was upregulated to 253.22 17.21% compared with control group. While, the caspase-9 activity was lowered to 150.10 11.36% relative to control after the pretreatment with catalpol (Fig. 6B).
Fig. 4. Western blot analysis of apoptotic protein. Showing Bax (21 kDa), cytochrome c (15 kDa) release into cytosolic fraction from the mitochondria, cleavage of pro-caspase-3 (32 kDa) into the active (20 kDa) form; cleavage of procaspase-9 (47 kDa) into the active forms. Reprobing for b-actin served as internal loading control. Western blot data are representative of three independent experiments with similar results.
4. Discussion In the present study, we found that exposure of cortical neurons to catalpol attenuated Ab1–42-induced apoptosis mainly via mitochondrial-dependent caspase cascade. The effects were visualized by using a wide range of different parameters including MTT cell viability and LDH cytotoxicity assay, ROS accumulation assay, mitochondrial membrane potential and cytochrome c determination, caspase-3 and caspase-9 analysis as well as morphological analysis in neuronal cells. Since Ab-induced apoptosis results in neurodegeneration, our results suggested that catalpol, isolated from the Chinese traditional herb R. glutinosa, might be potentially an anti-apoptotic agent against neurodegeneration. It is generally recognized that application of Ab to cultured cortical or hippocampal neurons mimics the pathogenesis of AD and Ab contributes to neurodegeneration in AD through activation of an apoptotic pathway (Carter, 2008). In our present study, the cortical neuron culture treated with 5 mM Ab1–42 for 72 h was served as AD cell model and the neuroprotective effect of catalpol on AD was investigated. First of all, it is well known that the progressive deterioration of hippocampal and cortical neurons emerges in brains of AD. Exposure to Ab1–42 resulted in an obvious damage in cortical neurons, which was visualized after immunocytochemical staining. Pretreatment with catalpol rescued cortical neurons from Ab1–42-induced neurotoxicity, which was revealed by morphological observation after immunocytochemical staining and also further confirmed by LDH cytotoxicity assay. The Bcl-2 family of proteins, an important endogenous regulator in cellular activity after a variety of physiological and pathological insults, has been suggested to be directly dependent on the elevation of Bax and its translocation to the mitochondrial
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Fig. 5. Effects of catalpol on the elevation of the accumulation of intracellular ROS, and the loss of mitochondria membrane potential due to Ab1–42 treatment. After treatment with 5 mM Ab1–42 in the presence or absence of 0.5 mM catalpol for 72 h, the three parameters were wholly assayed by fluorescent measurement. The changes of the three parameters were expressed as the percentage of control. (A). The production of intracellular ROS increased compared with control after Ab1–42 treatment and the administration of catalpol resulted in a dramatic decrease. (B). Catalpol significantly rescued mitochondrial membrane potential from dropping. Data were expressed as means S.E.M. of three independent experiments. *p < 0.05 compared with the 5 mM Ab1–42-treated cultures, ##p < 0.01 compared with control samples.
membrane (Kazmierczak et al., 2008). After translocated to the mitochondrial membrane, Bax can homodimerize and activate the terminal caspases by alteration of mitochondrial functions, resulting in the release of apoptosis promoting factors into the cytoplasm (Yuan and Yankner, 2000). Obviously, the protective effects of catalpol against Ab1–42 toxicity observed here are, at least in part, attributed to its ability to suppress up-regulation of the pro-apoptotic protein Bax. Our previous studies were consist with this result that Bax expression is reduced by catalpol in PC12 cells with an acute H2O2 treatment and catalpol modulates the expressions of Bax and attenuates apoptosis in gerbils after ischemic injury(Jiang et al., 2004; Li et al., 2006). Mitochondrial dysfunction, the excessive production of reactive oxygen species, and the decrease of mitochondrial membrane potential have long been implicated in the process of apoptosis (Zhang et al., 2007). It is generally accepted that the opening of mitochondrial permeability transition pore (MPTP) contributes to mitochondrial dysfunction, resulting in osmotic swelling of mitochondrial matrix, dissipation of the membrane potential, cessation of the ATP synthesis, the release of cytochrome c (Agostinho et al., 2008). ROS such as hydrogen peroxide, superoxide anion and hydroxyl radical readily damages biological molecules, ultimately results in apoptotic or necrotic cell death
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Fig. 6. Caspase-3 and caspase-9 activities in cortical neurons. Cultures were pretreated with catalpol (0.5 mM) for 30 min prior to 5 mM Ab1–42 for 72 h. After treatment, caspase-3, -9 activities were measured. The values were expressed as a percentage of caspase-3 or caspase-9 activities of control cultures and are the means S.E.M. of three independent experiments. *p < 0.05, **p < 0.01 compared with cultures exposed to 5 mM Ab1–42, ##p < 0.005 compared with control.
and is involved in the apoptotic mechanism of Ab-mediated neurotoxicity (Behl et al., 1994). The important role of mitochondria in apoptotic cell death has been reinforced by study showing the contribution of ROS in cell death signaling (Fleury et al., 2002). The overproduction of ROS, mainly produced by mitochondria, leads to oxidative stress and cellular oxidative pathways that proceed through apoptosis appear to be a predominant factor in the cell loss observed during Alzheimer’s disease (Chong et al., 2005). In the current study, Ab1–42 triggered significant accumulation of intracellular ROS as well as the overproduction of ROS further led to mitochondrial dysfunction, which ultimately resulted in apoptosis. Rising evidence confirmed that ROS is extensive in the AD brains, and plays a key role in Ab-induced neuronal cell death (Miranda et al., 2000). In agreement with the research, this study demonstrated that Ab1–42 induces ROS generation, and the intracellular ROS was scavenged by catalpol at 0.5 mM. As Somayajulu et al. (2005) proposed, mitochondrial control of apoptosis occurs at two levels, the maintenance of ATP production and mitochondrial membrane potential. The collapse of mitochondrial membrane potential was occurred in mitochondria level after Ab1–42-induced apoptosis, in agreement with previous study (Wang et al., 2001). Disruption of mitochondrial membrane potential by reactive oxygen species may release apoptosis-inducing factors which activate caspase cascade, cause nuclear condensation, and generate secondary reactive oxygen species (Petit et al., 1996). The release of cytochrome c from
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mitochondria to the cytosol is essential for caspase activation and cleavage. In the present study, we have shown that catalpol inhibited Ab1–42-induced cytochrome c release and the cleavage of caspase-3 and caspase-9. The activation of caspase-3 is also believed to be important for commitment to or execution of neuronal apoptosis (Harada and Sugimoto, 1999). The suppressive effect of catalpol on caspase-3 and caspase-9 activity further suggests that the protective effect of catalpol on cell death is related to its mitochondrial-dependent caspase pathway. In summary, catalpol attenuated neuronal apoptosis not only by reversing intracellular ROS accumulation, Bax level, mitochondrial dysfunction to some extent, but also through regulating the expression of caspase-3 and caspase-9. Thus, catalpol protects primary cultured cortical neurons induced by Ab1–42 through a mitochondrial-dependent caspase pathway. Taken together, catalpol effectively protects cortical neuronal cells from the Ab1–42induced neurotoxicity and suggest a neuroprotective effect on Ab1–42 insult. Acknowledgements This work was supported by grants from the Natural Science Foundation of Liaoning (20072185) and Science and Technology Project of Dalian (2008E11SF163). References Agostinho, P., Lopes, J.P., Velez, Z., Oliveira, C.R., 2008. Overactivation of calcineurin induced by amyloid-beta and prion proteins. Neurochem. Int. 52 (6), 1226– 1233. Allen, J.W., Eeldadah, B.A., Huang, X.L., Knoblach, S.M., Faden, A.I., 2001. Multiple caspases are involved in b-amyloid-induced neuronal apoptosis. J. Neurosci. Res. 65, 45–53. Behl, C., Davis, J.B., Lesley, R., Schubert, D., 1994. Hydrogen peroxide mediates amyloid bprotein toxicity. Cell 77, 817–827. Bi, J., Jiang, B., Hao, S., Zhang, A., Dong, Y., Jiang, T., An, L.J., 2009. Catalpol attenuates nitric oxide increase via ERK signaling pathways induced by rotenone in mesencephalic neurons. Neurochem. Int. 54, 264–270. Carter, C.J., 2008. Interactions between the products of the Herpes simplex genome and Alzheimer’s disease susceptibility genes: relevance to pathological-signalling cascades. Neurochem. Int. 52 (6), 920–934. Chong, Z.Z., Li, F., Maiese, K., 2005. Stress in the brain: novel cellular mechanisms of injury liked to Alzheimer’s disease. Brain Res. Rev. 49, 1–21. Fleury, C., Mignotte, B., Vayssiere, J.L., 2002. Mitochondrial reactive oxygen species in cell death signaling. Biochimie 84, 131–141. Harada, J., Sugimoto, M., 1999. Activation of caspase-3 in beta-amyloid-induced apoptosis of cultured rat cortical neurons. Brain Res. 842, 311–323. Hauptmann, S., Keil, U., Scherping, I., Bonert, A., Eckert, A., Muller, W.E., 2006. Mitochondrial dysfunction in sporadic and genetic Alzheimer’s disease. Exp. Gerontol. 41, 668–673. Jiang, B., Liu, J.H., Bao, Y.M., An, L.J., 2004. Catalpol inhibits apoptosis in hydrogen peroxide-induced PC12 cells by preventing cytochrome c release and inactivating of caspase cascade. Toxicon 43, 53–59.
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