J Biomed Sci 2003;10:379-388 DO I: 10.1159/000071157
Received: September 24, 2002 Accepted: March 24, 2003
IIIII II
Distinct Mechanisms Account for 13-Amyloid Toxicity in PC 12 and Differentiated PC12 Neuronal Cells
Y e n - J e n S u n g a C h i a - I o C h e n g c C h a i o - S u n g Chen b H s i e n - B i n H u a n g d
F o n g - L e e H u a n g a P e i - C h u n W u b M i n g - S h i S h i a o e H u e y - J e n T s a y b
alnstitute of Anatomy and Cell Biology, School of Medicine, and blnstitute of Neuroscience, School of Life Science, National Yang-Ming University, Taipei, CDepartment of Neurosurgery, Chi-Mei Foundation Hospital, Tainan, dlnstitute of Molecular Biology, National Chung Cheng University, Chia-Yi, eDepartment of Medical Research and Education, Veterans General HospitaI-Taipei, Taipei, Taiwan, ROC
Key Words Alzheimer's disease • 13-Amyloid • Apoptosis. Reactive oxygen species
Abstract Whether reactive oxygen species (ROS) mediate 13-amy- Ioid (AI3) neurotoxicity remains controversial. Naive PC12 cells (PC12) and nerve growth factor-differentiated PC12 cells (dPC12) were used to study the role of ROS in cell death induced by A~25-35. The viabil ity of PC12 and dPC12 cells decreased by 30-40% after a 48-hour expo- sure to 20 ~M A~25_35. Microscopic examination showed that A~25_35 induced necrosis in PC12 cells and apoptosis in dPC12 cells. Vitamin E (100 gM) and other antioxi- dants protected PC12 cells, but not dPC12 cells, against the cytotoxic effect of A~25-35. Since H20 2 has been pro- posed to be involved in A# toxicity, the effects of H20 2 on PC12 and dPC12 cells were studied. Differentiated PC12 cells appeared to be sgnificantly more resistant to H202 than naive PC12 cells. These data suggest that ROS may mediate A~25-35 toxicity in PC12 cells but not in dPC12 cells. Because the intracellular levels of ROS were ele-
vated during the differentiation of PC12 cells, the base- line levels of ROS in these two model cell types may determine the intracellular mediators for A~25_35 toxicity. Therefore, the protective effects of antioxidants against AI3 may depend upon the redox state of the cells.
Alzheimer's disease (AD) is a major neurodegenerative disease. The characteristic pathological features of AD are the presence of neurofibrillary tangles and senile plaques along with loss of neuronal cells. Various genetic altera- tions and mutations, including the amyloid precursor pro- tein, presenilin-1, presenilin-2, and apolipoprotein E, have been linked to familial forms of AD [14, 32]. The principal component in senile plaques is the 13-amyloid peptide (AI3), a peptide of 39-42 amino acids derived from the amyloid precursor protein by the proteolytic activities of 13-secretase and 7-secretase. The full-length A~ (A~1-42), the shorter peptide A[~l-40, and the 'toxic' fragment containing amino acid residues 25-35, A1325-35,
Dr. Huey-Jen Tsay Institute of Neuroscience, School of Life Science National Yang-Ming University, 155 Li-Nung Street, Section 2 Taipei 112, Taiwan (ROC) Tel. +886 2 28267154, Fax +886 2 28200259, E-Mail [email protected]
have been shown to induce neuronal death both in vitro and in vivo [2, 5, 6, 8, 15, 20, 21]. Numerous reports have suggested that extracellular deposition of A~ plays an important role in the pathogenesis of AD. The mecha- nism of Al3-induced neuronal cell death, however, re- mains controversial.
It has been postulated that the generation of oxidative stress is involved in AI3 toxicity [34, 40]. Exposure of neu- ronal cells to AI3 may lead to a disturbance in calcium homeostasis and impairment of mitochondrial function, both of which have been at tr ibuted to the product ion of reactive oxygen species (ROS) [ 1, 18, 21 ]. Local high con- centrations of ROS may cause functional alterations in lipids [23], which may result in the loss of fluidity of bio- logical membranes, including plasma membranes, endo-
plasmic reticulum, and mitochondrial membranes. The progressive loss of membrane fluidity can then lead to a further increase in the permeabili ty to Ca 2+ [7, 27]. In accordance with the hypothesis ofROS-media ted A[3 neu- rotoxicity [28, 29] is the fact that antioxidants and free radical scavengers have been shown to protect neuronal cells against AI3 toxicity [4, 24, 25, 33, 39, 44]. However, there have been conflicting data indicating that ROS are
not the pr imary cause of AI3 toxicity. It has been shown that while antioxidants reduce lipid peroxidation and ROS product ion induced by A[3, they do not prevent neu- ronal death under experimental conditions [16, 26]. Luc- ca et al. [17] further studied the protective effects of antioxidants against At3 in the presence and absence of serum and suggested that the potency of ROS to mediate AI3 toxicity was dependent, in part, upon the culture con- ditions of the cells. Because serum withdrawal can induce intracellular accumulation of ROS [t 7], we address in the present study the question of whether oxidative stress underlies A[3 neurotoxicity in PC12 neurons differen- tiated by nerve growth factor (NGF) under serum- deprived conditions. Our results show that ROS may mediate the cytotoxicity of A~25-35 in naive PC12 cells cultured in serum-supplemented conditions, but not in NGF-different iated PC 12 cells. Thus, the cellular level of ROS may play a differential role in the utilization of sig- naling pathways triggered by A~ as well as in the protec- tive effects of antioxidants against AI3 toxicity.
Materials and Methods
Reagents Murine NGF (mNGF 2.5S) was purchased fi'om Alomone (Jeru-
salem, Israel). Hoechst 33258, 2',7'-dichlorofluorescein diacetate (DCFDA), propidium iodide (PI), and hydroethidine (HE) were put-
chased from Molecular Probes (Eugene, Oreg., USA). Vitamin E was purchased from Sigma (St. Louis, Mo., USA). A[325-35 and the broad- spectrum caspase inhibitor, Z-VAD, were purchased from Bachem (Torrance, Calif., USA). The WST-1 (4-[3-(4-iodophenyl)-2-(4-nitro- phenyl)-2H-5-tetrazolio]-l,3-benzene disulfonate) reduction assay kit for cell viability was purchased from Roche Molecular Biochemi- cals (Mannheim, Germany).
Celt Culture aim Dtwg Treatrnent The PC12 rat pheochromocytoma cell line was purchased from
American Type Culture Collection (Manassas, Va., USA). Cells were grown in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum, 10% horse serum, and 100 gg/ml gentamycin at 37 ° C in a humidified incubator supplemented with 95% air/5 % CO2. Cells were seeded at a density of 5 x 103 cells/well on 96-well plates and 3 x 105 cells on 60-mm plates. For experiments with naive PC12 cells, fresh medium containing A1325-35 (or H202) was added 3 days after seeding. PC12 cells were differentiated with 50 ng/ml NGF in DMEM in the absence of serum. Four days after NGF addition, half of the volume of the medium was replaced with DMEM containing 50 ng/ml NGF and At3 or H202. Synthetic A1325-35 was prepared at a concentration of I rm'~f in water, and aggregated at 4°C for 60 h and at 37 °C for 8 h tbllowing the instructions &the manufacturer [22].
WST-1 Reduction Assay and Trypan Blue Exclusion Assay WST-1 is converted into water-soluble formazan by the redox
activity of living cells. Cell viability was measured with the WST-1 reduction assay following the instructions of the manufacturer. After treatment with A1325-35 for 24 or 48 h, 10 gl of diluted WST- 1 (diluted 1:1 with PBS) was added to the cells in a 96-well plate. After a 3-hour incubation at 37 ° C, the absorbance of water-soluble formazan was measured with a Multiskan RC ELISA plate reader (Thermo Labsys- terns, Vantaa, Finland) at a test wavelength of 450 nm and a refer- ence wavelength of 690 nm. To verify the cell viability measured by the WST-1 reduction assay, a t~'pan blue exclusion assay was employed. Briefly, after treatment with At325-35 and H202, cells on 60-mm plates were harvested and suspended in 0.4% trypan blue. Under a light microscope, viable cells appeared colorless and leaky cells appeared blue. Data were expressed as a percentage of colorless cells after treatment compared with the control sample.
Itoechst 33258 and PI Staining to Assess Apoptosis or Necrosis Induced by At3 Cells were grown on coverslips (10 mm in diameter) at 1.5 x 105
cells/ml in a 35-ram dish. At the end of cell culture, cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformalde- hyde in PBS for t5 rain at 4°C. To assess apoptotic cells, cells were incubated with 0.2% Triton X-100 for 10 rain at room temperature and then stained with 1 gg/ml Hoechst 33258 in PBS for 10 rain at room temperature. Apoptotic cells with condensed nuclei were exam- ined under UV illumination using a Nikon (Tokyo, Japan) E800M fluorescence microscope [42]. Necrotic cells which had lost the integ- rity of their plasma membranes were assessed by staining with PI to which non-necrotic cells are impermeable. PC12 cells in the growth medium were incubated with 10 gg/mt PI at 37°C for t0 min. PI- positive cells were observed at 525 nm using a Nikon E800M fluores- cence microscope [371.
Measurement of H202 and the Superoxide Anion (02) The intracellular level of H202 in PC 12 cells during differentia-
tion was measured using DCFDA [25, 30]. Plasma membranes are freely permeable to the diacetate ester, DCFDA; however, once it enters a cell, it is hydrolyzed by cytosolic esterases to 2',7'-dichloro- fluorescin (DCF), which reacts with peroxide to form the fluorescent 2',7"-dichlorofluorescein. Cells were incubated in 30 gM DCFDA for 30 rain at 37 °C and rinsed twice with PBS. The fluorescence intensi- ty of 2',7'-dichlorofluorescein was then observed using a Nikon ES00M fluorescence microscope, or quantified by flow-cytometric analysis (Becton-Dickinson, Bedford, Mass., USA). To measure the intraceltular levels of O z, cells were loaded with I 0 uM HE for 30 min at 37 ° C [35]. HE is oxidized by O~ to form ethidium, which interca- lates into nucleic acids and fluoresces at 590 nm upon 465-nm excita- tion. The intracellular production of O~ was examined under a fluo- rescence microscope, and the level was quantified by flow-cytometric analysis (Becton-Dickinson). The excitation wavelength used by flow cytometry was set at 488 nm. The observation wavelength was set at 530 nm for DCFDA staining and 585 nm for HE staining. The emit- ted fluorescence was collected on FL1 and FL2 channels, respectively [31]. Data were acquired and analyzed using Cell Quest software (Becton-Dickinson).
Statistics Data are expressed as means +_ SEM. Statistical significance was
analyzed with the SAS program on a Pentium-III-based personal computer using one-way ANOVA followed by the LSD test. All dif- ferences of p < 0.05 were considered significant.
Results
A~25_35-Induced Cell Death in Naive PC12 and NGF-D~fferentiated PC12 (dPC12) Cells The cytotoxic effects of A[325-35 on naive PC12 and
dPC12 cells were first examined. The WST-1 reduction assay showed that control naive PC 12 cells actively prolif- erated up to 48 h after seeding with more than a 2-fold increase in WST- 1 reduction on day 2 as compared to day 1. In contrast, :no significant changes in WST-1 reduction by dPC12 cells were observed between days 1 and 2 due to NGF-induced differentiation, and therefore, they are con- sidered to be quiescent. Cell viability of both naive PC 12 and dPC12 cells decreased by 20 and 30-40% following incubation with 20 gM A[325-35 for 24 and 48 h, respec- tively (fig. 1 a, b). Staining of A[325_35-treated dPC 12 cells with Hoechst 33258 revealed characteristic patterns of apoptotic nuclei with condensed chromatins. Interesting- ly, such condensed chromatins were rarely found in naive PC12 cells exposed to A~25-35, despite the significant decrease in WST-1 reduction (fig. lc).
Previous studies have shown that the apoptotic path- way involves activation of the caspase cascade [ 10, 24, 38, 41]; therefore, we examined the protective effects of a
broad-spectrum caspase inhibitor, z-VAD, on A~25-35-
treated dPC12 cells by adding 40 pM z-VAD together with 20 gMA[~25-35 into dPC12 cells for 48 h. As shown in figure 2a, the cell viability of dPC 12 cells after treatment with 20 gMA[~25-35 alone for 48 h was 49 + 1.9°/0 (rela- tive to the day 1 control). In the presence of z-VAD, how- ever, the cell viability of A[325_35-treated dPC 12 cells was significantly elevated to 89.9 + 2.5%. The addition of z- VAD to dPC12 cells in the absence of A~25-35 also pro- moted dPC 12 cell viability to 114.8 + 6.6 %. The number of apoptotic nuclei (nuclei with chromatin condensation) was also significantly reduced by the addition of z-VAD to A1325_35-treated dPC12 cells (fig. 2b).
Because condensed nuclei were not found among A[325_35-treated naive PC12 cells, necrotic pathways in these cells were further examined by PI staining. After A1325-35 treatment for 48 h, numerous PC12 cells were found to be PI positive (fig. 3a). Since decreases in WST-1 reduction in A1325_35-treated PC12 cells could be due to growth inhibition or cell death, the trypan blue exclusion assay was used to enumerate deaths of PC 12 cells induced by A~25-35. AS shown in figure 3b, A[325-35 caused a signifi- cant decrease in the percentage of viable PC12 cells. The reverse peptide, A[~35-25, showed no toxic effect on either PC12 or dPC12 cells (data not shown).
Differential Role of ROS in AB Cytotoxicity in Naive PC12 and dPCI2 Cells Since oxidative stress has been proposed to be a major
mechanism of A[3 cytotoxicity, the contribution of ROS to the cytotoxicity of AI325-35 in naive PC 12 and dPC 12 cells was examined using an antioxidant, such as vitamin E, that has been shown to attenuate the deleterious effects of A[3 [3, 26, 39]. Vitamin E (100 gM) was added to PC12 and dPC 12 cells in the presence and absence of 20 ~t~I A[3 for 48 h, and cell viability was measured by the WST-1 reduction assay. The addition of vitamin E alone to the cell culture appeared to have no effect on the viability of naive PC12 cells in the absence ofA[325_35 (100.1 + 7.4%, relative to control PC 12 cells with no treatment); how- ever, vitamin E significantly promoted the viability of PC12 cells treated with A[325_35 (82.1 _+ 5.1 vs. 62.4 + 5.1% of the control, for cells with and without vitamin E addition, respectively; p < 0.01; fig. 4, dark bars). On the other hand, vitamin E had no detectable effects on the cell viability of dPC12 cells in the presence or absence of A[~25-35 (58.1 --- 5.0 vs. 61.0 -+ 5.0% for A[3-treated dPC 12 cells with or without vitamin E addition, respec- tively, and 95.1 + 3.0% for dPC12 cells treated with vita- min E alone; fig. 4, open bars). Hoechst 33258 staining for
Differential Pathways Mediate AI3 Neurotoxicity
J Biomed Sci 2003; 10:37%388 381
Fig. 1. Cytotoxicity and changes in nuclear morphology of A[~25_35-treated PC12 and dPC12 cells, n, b Cell viability measured by the WST-1 reduction assay in PC12 (a) and dPC12 cells (b) incubated with 20 pM A~325_35 for 24 and 48 h (n = 4, * p < 0.05, • *p < 0.01). e Hoechst 33258 staining for illustrating apoptotic cells in the absence and presence of A~25-35. Apoptotic cells con- tained condensed chromatin and/or frag- mented DNA; therefore, nuclei appeared smaller and brighter in staining intensity or were fragmented (arrows indicate several, but not all, apoptotic nuclei as examples).
a
250
g ~. 200
o 150
100 7-. s
5o
"O
Vehicle 20 gMAI3
b 150
go '~ g loo
(D >.
co ~5 50 ~ g
v
m Vehicle 20 g M A ~
24 h 48 h 24 h 48 h PC12 dPC12
Vehicle AI325_35
chromatin condensation also confirmed that vi tamin E treatment had no effect on the percentage of apoptotic cells in dPC 12 cells treated with A~25-35 (data not shown). Furthermore, NAC, catalase, and propyl gallate had no effect on the cell viability or apoptosis of A1325_35-treated dPC 12 cells (data not shown).
The data presented in figure 4 established a distinct effect of antioxidants on the viability of naive and differ- entiated PC12 cells following A[3 exposure. It has also been demonstrated that multiple pathways may mediate AI3 toxicity [41]. Therefore, we further studied the possi- ble mechanisms leading to the differential protective
382 J B i o m e d Sci 2003; 1 0 : 3 7 9 - 3 8 8 Sung/Cheng/Chen/Huang/Huang/Wu/ Shiao/Tsay
Fig. 2, Effect of z-VAD on AI325_35-treated dPCI2 cells, a dPC12 cells incubated in the presence or absence of 40 gM z-VAD in combination with of 20 btM AI325-35 for 48 h. Cell death was quantified by the WST-1 reduction assay (n = 4, **p < 0.01). b Apoptotic nuclear morphology of A[325_;5-treated dPC 12 cells reduced by z-VAD.
a
140
~ 1 2 0 '~ o
• ~ 100
~ 8 80
~ 40 2O
AI3 Ap + z-VAD z-VAD
effect of antioxidants on A[325_35-treated PC 12 and dPC 12 cells. While involvement of ROS in the toxicity of A[3 has been widely accepted, BeN et al. [3] demonstrated that the role of ROS, e.g. H202 production induced by A}, in A[3-mediated toxicity may be distinct among different cell types. We compared the sensitivity of PC 12 and dPC12 cells to the extracellular addition of H202, which is freely permeable in plasma membranes. We then examined the level of intracellular H202 of PC12 and dPC12 cells before and after the addition of extracellular H202. A sig- nificant increase in intracellular H202, as measured by flow-cytometric analysis, was found in both PC12 and dPC12 cells treated with 40 gMH202 for 15 min (fig. 5a, b), indicating that extracellular H202 penetrated through the membrane of PC 12 and dPC 12 cells. Treatment with H202 appeared to be cytotoxic to both PC12 and dPC12 cells, since concentration-dependent cell death kinetics was observed in these cells following the addition of 20, 40, 80, and 100 gM H202 (fig. 5c). However, PC12 cells were significantly more sensitive to H202 than were dPC12 cells, because 40% cell death was found in naive PC 12 cells with 40 btM H202 treatment, yet a comparable degree of cell death in dPC 12 cells was only reached with 100 ~MH202 (fig. 5c).
We postulated that the different sensitivities of PC 12 and dPC12 cells to H202 may have been due to the dis- tinct intracellular levels of ROS in these two cell systems. To examine this hypothesis, we measured the intracellu- lar levels of ROS in these cells using DCFDA and HE, which fluoresce after reacting with H202 and 02, respec- tively. The fluorescence intensities measured by using these two dyes significantly increased during differentia- tion of PC12 cells; levels of different intensity were detected by both fluorescence microscopy (fig. 6a-f) and flow cytometry (fig. 6g, h). The basal level of 0 2 that is constitutive to naive PC12 cells (fig. 6d) was increased by leakage of O~ from the active mitochondrial electron transport system. In contrast, no H202 (DCF) fluores- cence was detectable before the induction of differentia- tion (fig. 6a). Intraceltular levels of both H202 and O~ accumulated significantly within 1 day after NGF treat- ment in combination with serum deprivation (fig. 6b, e and the pink line in fig. 6g, h). Levels of H202 and O~ remained elevated for at least 4 days after the induction of differentiation by NGF and serum withdrawal, confirm- ing that intracellular levels of ROS in dPC 12 cells were elevated as compared with those of naive PC 12 cells.
Differential Pathways Mediate A~ Neurotoxicity
J Biomed Sci 2003; 10:379-388 383
a Phase con t ras t PI f l uo rescence
Fig. 3. Detection of necrotic cells and viable cell counts, a Naive PC12 and dPC12 cells cultured in 96-well plates with 20 ~tM A13zs-35 treatment for 24 h. Necrotic cells were detected by staining with PI and obser- vation under a fluorescence microscope. Bar = 50 gin. b Trypan blue exclusion assay performed to enumerate viable cells among PC12 cells treated with 20 ~tM A1325-a5 for 24 h.
b
120
100
.=_- .b 80
...Q t-" 0 • .~ ~ 60
= o 40 8~ 20 i
Cont ro l A~
D i s c u s s i o n
It is well documented that A~ plays a central role in the pathogenesis of AD. However, mechanisms mediating A[~ toxicity are still not fully understood. Results from the present report suggest that the degree to which ROS mediate the cytotoxicity of AI3 and their potency depend upon the types and redox states of the cells involved. We
found that A~25-35 induced necrosis in PC12 cells and apoptosis in dPC12 cells. The antioxidant, vitamin E, protected only PC12 cells, but not dPC12 cells, against AI325_35-induced cytotoxicity. On the other hand, the broad-spectrum caspase inhibitor, z-VAD, effectively re- duced the percentage of apoptotic ceils and promoted cell viability in dPC 12 cells. NGF treatment and serum depri- vation of PC12 cells caused significant increases in intra-
Fig. 4. Effect of vitamin E on A[325-35- treated PC12 and dPC12 cells. PC12 and dPC12 cells were incubated in the presence or absence of 100 ~tM vitamin E for 48 h with or without 20 gM At]25-35 treatment. Cell viability was quantified by the WST-1 reduction assay. **p < 0.01.
120
80 g
"o
"7 1-
40-
PC12 cells dPC12 cells
A~ A~ + Vitamin E Vitamin E
Fig. 5. Effect ofextracellular H202 on PC12 and dPCI2 cells. PC12 (a) and dPC12 cells (b) incubated with 40 ~tM H20; for 15 min, then loaded with DCFDA and analyzed by flow cytometry assay, c PC12 and dPC12 cells incubated with 20, 40, 80, and 100 gM H202 for 24 h. Cell death was quantified by the trypan blue exclusion assay,
(For fig. 6 see page 386) Fig. 6. Accumulation of ROS during differentiation of PC 12 cells, a - f PC 12 cells incubated with 50 ng/ml NGF plus serum deprivation for different periods of time (days 0-2). Cells were loaded with DCFDA (green) and HE (red) and observed under a fluorescence microscope. Bar = 20 gm. g, h PC12 cells incubated with NGF in serum-free medium for 1-4 days, respectively, then loaded with DCFDA (g) and HE (h). The fluorescence intensity was analyzed by flow cytometry.
cellular levels of H202 and O i that remained elevated even 4 days after NGF treatment and serum withdrawal. Therefore, it is likely that the elevated baseline level of ROS in dPC 12 cells may confer resistance to these cells against the toxic effect of H202. Furthermore, our data support distinct pathways being utilized by Al3-triggered toxic signals in cells with different intracellular levels of ROS.
The fact that A[3 caused lipid peroxidation and accu- mulation of intracellular H202 indicates the association of A~3 toxicity with oxidative stress in both cultured cells and brains of AD patients [11, 36]. However, there are contra- dictory results indicating that free radicals might not mediate A~ toxicity [17, 43]. Although lipid peroxidation induced by AI3 can be inhibited by antioxidants, the cell viability of rat primary cortical and hippocampus neu- rons was not increased by antioxidant treatment [ 16, 26]. Lockhart et al. [16] also showed that free radical scaven- gers, including vitamin E, glutathione, and catalase, failed to attenuate the toxicity of A[3. Therefore, multiple path- ways, other than ROS, have been proposed which me- diate the toxic signal of AI3, including mitochondria and receptors [ 17, 41 ]. Furthermore, several studies have sug- gested that H202 and AJ3 may cause cell damage through different pathways. For example, glyceraldehyde-3-phos- phate dehydrogenase activity and glucose oxidation have been shown to be modulated by H202, but are not influenced by AI3 treatment [43]. White et al. [42] have also shown that the expression of the amytoid precursor- like protein 2 was induced in A~-treated cortical neurons but not in H202-treated neurons.
Several possibilities underlie the inconsistency in the role of ROS and the protective effects of antioxidants against A[3 toxicity in the two different model cell types used in this study. Baseline levels of intracellular ROS in naive and differentiated PC 12 cells may influence the cel- lular response to oxidative stress induced by A~3. It has been shown that pretreatment with oxidants at a tow con- centration can prevent H202-induced neuronal cell death under certain conditions [12]. We have shown that the baseline level of ROS in naive PC 12 cells was significantly lower than that of dPC 12 cells. This may account for the higher sensitivity of PC12 cells to H202 compared to dPC 12 cells. Lucca et al. [17] investigated the influence of culture conditions on the protective effects of antioxi- dants against A~3 and found that antioxidants protected primary cortical cells from A~ insult only in the presence of fetal calf serum. The fact that serum withdrawal can induce ROS production and cell death in primary cells and PC12 cells [9, 13, 19] lends consistency to the results of Lucca et al. [17] and the present study. We propose that differential residual or baseline levels of ROS in PC12 cells and dPC 12 cells may be the determining factors for the significance of ROS in mediating At3 toxicity and for the effectiveness of antioxidant protection.
Acknowledgments
This study was supported in part by research grant CMYM 9003 from the Chi-Mei Foundation, VGH89-382-5 from Veterans Gener- al Hospital-Taipei, and 90-B-FA22-1-4-02 from the Ministry of Edu- cation. We thank Dr. Lang-Sian Kao and Dr. Young-Ji Shiao for crit- ically reviewing the manuscript.
2 Behl C, Davis JB, Klier FG, Schubert D. Amy- loid 13 peptide induces necrosis rather than apoptosis. Brain Res 645:253-264; 1994.
3 Behl C, Davis JB, Lesley R, Schubert D. Hy- drogen peroxide mediates amyloid 13 protein toxicity. Cell 77:817-827; 1994.
4 Cafe C, Torri C, Bertorelli L, Angeretti N, Luc- ca E, Forloni G, Marzatico F. Oxidative stress after acute and chronic application of 13-amy- loid fragment 25-35 in cortical cultures. Neu- rosci Lett 203:61-65; 1996.
5 Cotman CW, Anderson AJ. A potential role for apoptosis in neurodegeneration and Alzhei- mer's disease. Mol Neurobioi 10:19-45;1995.
6 Cotman CW, Su JH. Mechanisms of neuronal death in Alzheimer's disease. Brain Pathol 6: 493-506; 1996.
7 Ekinci FJ, Malik KU, Shea TB. Activation of the L voltage-sensitive calcium channel by mi- togen-activated protein (MAP) kinase follow- ing exposure of neuronal celIs to I~-amyIoid. MAP kinase mediates ~3-amyloid-induced neu- rodegeneration. J Biol Chem 274:30322- 30327;1999.
8 Estus S, Tucker HM, van Rooyen C, Wright S, Brigham EF, Wogulis M, Rydel RE. Aggre- gated amyloid-J3 protein induces cortical neu- ronal apoptosis and concomitant 'apoptotic' pattern of gene induction. J Neurosci 17:7736- 7745; 1997.
9 Funakoshi H, Yonemasu T, Nakano T, Matu- moto K, Nakamura T. Identification of Gas6, a putative ligand for Sky and Axl receptor tyro- sine kinases, as a novel neurotrophic factor for hippocampal neurons. J Neurosci Res 68:150- 160;2002.
10 Giovanni A, Keramaris E, Morris E J, Hou ST, O'Hare M, Dyson N, Robertson GS, Siack RS, Park DS. E2F1 mediates death of f3-amyloid- treated cortical neurons in a manner indepen- dent of p53 and dependent on Bax and caspase 3. J Biol Chem 275:11553-11560;2000.
11 Good PF, Werner P, Hsu A, Olanow CW, Perl DP. Evidence of neuronal oxidative damage in Alzheimer's disease. Am J Pathol 149:21-28; t996.
12 Isacson O, Sco H, Lin L, Albeck D, Granholm AC. Alzheimer's disease and Down's syn- drome: Roles of APP, trophic factors and ACh. Trends Neurosci 25:79-84;2002.
Differential Pathways Mediate A~ Neurotoxicity
J Biomed Sci 2003;10:379-388 387
13 Kim HY, Akbar M, Lau A, Edsall L. Inhibition of neuronal apoptosis by docosahexaenoic acid (22:6n-3). Role of phosphatidylserine in anti- apoptotic effect. J Biol Chem 275:35215- 35223;2000.
14 Li J, Lee JM, Johnson JA. Microarray analysis reveals an antioxidant responsive element- driven gene set involved in conferring protec- tion from an oxidative stress-induced apoptosis in IMR-32 Cells. J Biol Chem 277:388-394; 2001.
15 Li YP, Bushnell AF, Lee CM, Perlmutter LS, Wong SK. 13-Amyloid induces apoptosis in hu- man-derived neurotypic SH-SY5Y cells. Brain Res 738:196-204; 1996.
16 Locldaart BP, Benicourt C, Junien JL, Privat A. Inhibitors of free radical formation fail to at- tenuate direct 13-amyloid25_35 peptide-me- diated neurotoxicity in rat hippocampal cul- tures. J Neurosci Res 39:494-505; 1994.
t 7 Lucca E, Angeretti N, Forloni G. Influence of cell culture conditions on the protective effect of antioxidants against [3-amyloid toxicity: Studies with lazaroids. Brain Res 764:293- 298;1997.
18 MacManus A, Ramsden M, Murray M, Hen- derson Z, Pearson HA, Campbell VA. En- hancement of 45Ca 2+ influx and voltage-depen- dent Ca 2+ channel activity by L3-amyloid (1-40) in rat cortical synaptosomes and cultured corti- cal neurons. Modulation by the proinflamma- tory cytokine interleukin- 113. J Biol Chem 275: 4713-47 t 8;2000.
19 Magnani E, Bettini E. Resazurin detection of energy metabolism changes in serum-starved PC 12 cells and of neuroprotective agent effect. Brain Res Brain Res Protoc 5:266-272;2000.
20 Markesbery WR. Oxidative stress hypothesis in Alzheimer's disease. Free Radie Biol Med 23:134-147;1997.
21 Mattson MP, Partin J, Begley JG. Amyloid [3- peptide induces apoptosis-related events in synapses and dendrites. Brain Res 807:167- 176;1998.
22 Murphy GM Jr, Yang L, Cordell B. Macro- phage colony-stimulating factor augments [3- amyloid-induced interleukin-1, interleukin-6, and nitric oxide production by microglial cells. J Biol Chem 273:20967-20971; 1998.
23 NitschRM, BlnsztajnJK, PittasAG, SlackBE, Growdon JH, Wurtman RJ. Evidence for a membrane defect in Alzbeimer disease brain. Proc Natt Acad Sci USA 89:1671-1675;1992.
24 Pappolla MA, Sos M, Omar RA, Bick RJ, Hickson-Bick DL, Reiter RJ, Efthimiopoulos S, Robakis NK. Melatonin prevents death of neuroblastoma cells exposed to the Alzheimer amytoid peptide. J Neurosci 17:1683-1690; 1997.
25 Pereira C, Santos MS, Oliveira C. Involvement of oxidative stress on the impairment of energy metabolism induced by A13 peptides on PC12 cells: Protection by antioxidants. Neurobiol Dis 6:209-219; 1999.
26 Pike CJ, Ramezan-Arab N, Cotman CW. t3- Amyloid neumtoxicity in vitro: Evidence of oxidative stress but not protection by antioxi- dants. J Neumchem 69:1601-1611;1997.
27 Praprotnik D, Smith MA, Richey PL, Vinters HV, Perry G. Plasma membrane fragility, in dystrophic neurites in senile plaques of Alz- heimer's disease: An index of oxidative stress. Acta Neuropathol (Berl) 91:1-5; 1996.
28 Pratico D, Uryu K, Leight S, Trojanoswki JQ, Lee VM. Increased lipid peroxidation precedes amyloid plaque formation in an animat model ofAlzheimer amyloidosis. J Neurosci 21:4183- 4187;2001.
29 Raina AK, Takeda A, Nunomura A, Perry G, Smith MA. Genetic evidence for oxidative stress in Alzheimer's disease. Neurorepol-~ 10: 1355-t357;1999.
30 Rothe G, Valet G. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2',7'-dichlorofluorescin. J Leukoc Bio147:440-448; 1990.
31 Satoh T, Sakai N, Kubo T, Enokido Y, Uchiya- ma Y, Hatanaka H. Flow cytometric analysis of serum deprivation-induced apoptosis of PC 12 cells, with special reference to role of bcl-2. Neurosci Lett 201:119-122;1995.
33 Simonian NA, Coyle JT. Oxidative stress in neurodegenerative diseases. Annu Rev Phar- macol Toxicol 36:83-106; 1996.
34 Smith MA, Rottkamp CA, Nunomura A, Rai- na AK, Perry G. Oxidative stress in Alzhei- mer's disease. Biochim Biophys Acta t502: 139-144;2000.
35 Sohal RS, Dubey A. Mitochondrial oxidative damage, hydrogen peroxide release, and aging. Free Radic Biol Med 16:621-626; 1994.
36 Subbarao KV, Richardson JS, Ang LC. Autop- sy samples of Alzheimer's cortex show in- creased pemxidation in vitro. J Neurochem 55: 342-345; 1990.
37 Tan J, Town T, Placzek A, Kundtz A, Yu H, Mutlan M. Bct-XL inhibits apoptosis and ne- crosis produced by Alzheimer's beta-amy- loidl-40 peptide in PC12 cells. Neurosci Lett 272:5-8;1999.
38 Troy CM, Rabacchi SA, Friedman WJ, Frap- pier TF, Brown K, Shelanski ML. Caspase-2 mediates neuronal cell death induced by 13- amyloid. J Neurosci 20:1386-1392;2000.
39 Ueda K, Shinohara S, Yagami T, Asakura K, Kawasaki K. Amyloid [3 protein potentiates Ca 2+ influx through L-type voltage-sensitive Ca 2+ channels: A possible involvement of free radicals. J Neurochem 68:265-271; 1997.
40 Varadarajan S, Yatin S, Aksenova M, Butter- field DA. Review: Alzheimer's amyloid [3-pep- tide-associated free radical oxidative stress and neurotoxicity. J Struct Biol 130:184-208; 2000.
41 Wang CN, Chi CW, Lin YL, Chen CF, Shiao YJ. The neuroprotective effects of phytoestro- gens on amyloid 13 protein-induced toxicity are mediated by abrogating the activation of cas- pase cascade in rat cortical neurons. J Biol Chem 276:5287-5295;200 I.
42 White AR, Zheng H, Gatatis D, Maher F, Hesse L, Multhaup G, Beyreuther K, Masters CL, Cappai R. Survival of cultured neurons from amyloid precursor protein knock-out mice against Alzheimer's amyloid-13 toxicity and oxidative stress. J Neurosci 18:6207-6217; 1998.
43 Zhang Z, Rydel RE, Drzewiecki GJ, Fuson K, Wright S, Wogulis M, Audia JE, May PC, Hys- lop PA. Amyloid [3-mediated oxidative and metabolic stress in rat cortical neurons: No direct evidence for a role for H202 generation. J Neurochem 67:1595-1606; 1996.
44 Zhou Y, Gopalakrishnan V, Richardson JS. Actions of neurotoxic [3-amyloid on calcium homeostasis and viability of PC12 cells are blocked by antioxidants but not by calcium channel antagonists. J Neurochem 67:1419- 1425;1996.
