Aluminum-Maltolate Induces Apoptosis and Necrosis in Neuro-2a Cells: Potential Role for p53 Signaling

Victor J. Johnson*,1, Sang-Hyun Kim{dagger} and Raghubir P. Sharma*,2

* Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia 30602, and {dagger} Research Center for Resistant Cells, College of Medicine, Chosun University, Gwangju 501-759, South Korea

Received September 21, 2004; accepted November 1, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aluminum maltolate (Al-malt) causes neurodegeneration following in vivo exposure, and apoptosis plays a prominent role. The objective of this study was to define the form of cell death induced by Al-malt and to establish an in vitro model system amenable to mechanistic investigations of Al-malt-induced cell death. Neuro-2a cells, a murine neuroblastoma cell line, were treated with Al-malt for 24 h, following which mode of cell death and alterations in apoptosis-related gene expression were studied. Al-malt concentration-dependently increased cell death. The mode of cell death was a combination of apoptosis and necrosis. Treatment with Al-malt resulted in caspase 3 activation and the externalization of phosphatidyl serine, both indicative of apoptosis. In addition, nuclear condensation and fragmentation were evident. Interestingly, pretreatment with cycloheximide (CHX), a potent protein synthesis inhibitor markedly reduced Al-malt-induced apoptosis, indicating that altered gene expression was critical for this form of cell death. Pretreatment with CHX had no effect on necrosis induced by Al-malt. Analysis of gene expression showed that p53 mRNA was increased following treatment with Al-malt. This increase was accompanied by a marked inhibition of Bcl2 expression and an increase in BAX expression, a pattern of gene expression suggestive of a pro-apoptotic shift. Results show for the first time that p53 is induced by Al in neuron-like cells and suggest that the p53-dependent intrinsic pathway may be responsible for Al-induced apoptosis. Future studies investigating the role of p53 in Al neurotoxicity both in vivo and in vitro are warranted.

Key Words: aluminum; maltolate; apoptosis; necrosis; caspase 3; neurodegeneration; neurotoxicity; Neuro-2a; p53; Bcl2; BAX.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The etiology of Alzheimer's disease (AD) is exceedingly complex, with contributing factors including oxidative stress (Pratico et al., 1998Go, 2002Go), inflammation (Akiyama et al., 2000Go) and extensive protein and membrane modification (Mattson et al., 2002Go). All of these pathophysiological processes are also associated with non-disease aging. It is not clear why some patients develop AD and others remain healthy into advanced age. Two major contributing factors in the development of AD are the existence of a genetic predisposition (Combarros et al., 2002Go) and exacerbation of normal aging-related changes by environmental factors (Mattson et al., 2002Go). Aluminum (Al) represents one such environmental factor that has been linked to the development of AD. In fact, it has been stated that the use of Al as an experimental neurotoxicant has recapitulated virtually every feature of the neurodegenerative spiral afflicting Alzheimer's patients (Strong, 2002Go). Additional evidence suggests that Al accumulates in the human brain with aging (Markesbery et al., 1981Go). Given that Al is the third most abundant element in the earths crust and exposure to it is unavoidable, further research into the neurotoxicity of this metal is both warranted and essential.

Aluminum maltolate (Al-malt) is a lipophilic complex of aluminum that forms a metastable solution of pH 7. This Al complex is advantageous for use in in vitro mechanistic studies because it dose not form insoluble precipitates of Al-hydroxide at physiological pH, as do most other salt forms of Al (Martin, 1991Go). Maltolate is also a common component of the human diet. It is a by-product formed during sucrose pyrolysis or thermal degradation of starch (Johnson et al., 1969Go) and can be found in coffee, soybean, baked cereals, and caramelized and browned foods (Gralla et al., 1969Go). Given the extremely high affinity of maltolate for Al, there is a potential for Al-malt to form in the gastrointestinal tract. Therefore, investigating the enhanced toxicity of Al-malt is relevant to human health. Numerous studies have demonstrated that Al-malt is a potent neurotoxin. Intracisternal injection of Al-malt produced cytoskeletal alterations (Klatzo et al., 1965Go; Savory et al., 1996Go, 1998Go) that were reminiscent of the tangles seen in AD. There is also evidence to suggest that maltolate may facilitate the entry of Al into the brain (van Ginkel et al., 1993Go), thereby increasing the potential for neurotoxicity. Recent findings show that the neurofibrillary degenerations induced by Al also contain hyperphosphorylated tau proteins as in AD (Savory et al., 1998Go). Increasing evidence suggests that oxidative stress and apoptosis are key events in the neuropathological changes following exposure to Al-malt (Savory et al., 1999Go).

Al-malt-induced apoptosis in the rabbit hippocampus involves perturbation of the Bcl-2/Bax ratio (Savory et al., 1999Go). More recently, it was shown that the endoplasmic reticulum is involved in Al-malt-induced apoptosis (Ghribi et al., 2001aGo). Translocation of Bax to the endoplasmic reticulum and a loss of Bcl-2 have been demonstrated in rabbits treated with Al-malt. These effects may be involved in the observed release of cytochrome c from the mitochondria in Al-malt-treated rabbits (Ghribi et al., 2002aGo). Once released, cytochrome c can bind to Apaf-1 in the cytoplasm, forming a complex that can activate caspase 9 with subsequent activation of death-inducing caspase 3 (Li et al., 1997Go). Ghribi et al. (2001a)Go demonstrated that caspase 3 was activated following intracisternal injection of Al-malt, and the site of activation was the endoplasmic reticulum (Ghribi et al., 2001aGo). These biochemical changes were shown to culminate in DNA fragmentation, as evidenced by terminal uridine nucleotide end labeling (TUNEL)-staining in the hippocampus of Al-malt-treated rabbit (Ghribi et al., 2002bGo; Savory et al., 1999Go). Collectively, these studies provide evidence for the involvement of apoptotic cell death in Al-induced neurodegeneration and strengthen the possible involvement of Al as an early factor in the development of AD. The pathway responsible for the observed shift in Bcl2/BAX ratio remains to be identified. One likely candidate is p53, which is known to differentially control expression of Bcl2 and BAX as well as other important genes involved in apoptosis.

Neuronal cell lines are also sensitive to the toxic effects of Al-malt and may provide models to study the mechanisms involved in apoptosis. Al-malt-induced apoptosis has been demonstrated in differentiated PC-12 cells (Ohyashiki et al., 2002Go; Tsubouchi et al., 2001Go) and human NT2 neuroblastoma cells (Griffioen et al., 2004Go). Neuro-2a (N2a) murine neuroblastoma cells are also sensitive to Al treatment, which up-regulated the expression and accumulation of neurofibrillay tangle protein (Abreo et al., 1999Go). This effect was associated with increased uptake of iron in N2a cells, possibly contributing to Al-induced oxidative stress and neurotoxicity. The objective of the present study was to develop an in vitro model that can be used to define the nature of Al-malt-induced cell death, apoptosis versus necrosis. The results clearly demonstrate the coinvolvement of apoptosis and necrosis in Al-malt neurotoxicity. The apoptotic cell death induced by Al-malt was dependent on de novo protein synthesis as evidenced by inhibition of apoptosis in the presence of cycloheximide (CHX). Gene expression studies showed altered expression of p53 and downstream signaling molecules, including Bax and Bcl-2, potentially implicating the p53 pathway in Al-malt-induced apoptosis. The present model will be useful in defining the pathways and mechanisms involved in Al-induced neurotoxicity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. Cell culture reagents were obtained from GIBCO Life Technologies, Inc. (Grand Island, NY). Annexin V-FITC was procured from Pharmingen (BD Biosciences Pharmingen, San Diego, CA). The CaspACETM assay system was purchased from Promega Corporation (Madison, WI). Propidium iodide (PI) and CHX were purchased from Sigma Chemical Company (St. Louis, MO). All other reagents used were purchased from Sigma and were tissue culture grade.

Cell culture and treatment. Murine neuroblastoma Neuro-2a (N2a; CCL-131, American Type Culture Collection, Manassas, VA) cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS) and 1% penicillin-streptomycin (Pen-Strep®, GIBCO, Grand Island, NY). Cultures were maintained in plastic tissue culture vessels in a humidified atmosphere at 37°C with 5% CO2. Cells were seeded at 2 x 105/ml for each experiment and allowed to grow for 18 h prior to treatment with Al-malt. All treatments were performed in DMEM with 10% FCS for duration of 24 h unless stated otherwise.

The toxicity of both Al3+ and Al-malt was investigated in the present study. The speciation chemistry of Al is very complex, and aqueous solutions of Al3+ undergo extensive hydrolysis over time, yielding colloidal complexes that may influence the bioavailability of Al (Campbell et al., 1999Go). To control this, Al3+ stocks were made immediately prior to treatment by dissolving AlCl3 in deionized water at a concentration of 10 mM, and this stock was used to make the treatment solutions (1–1000 µM AlCl3). The pH of the treatment solutions was adjusted to 7 prior to addition to the cells. Several dietary ligands, including citrate, lactate, and maltolate, have a high affinity for Al and can influence both the solubility of the metal and its toxicity. Numerous studies have demonstrated that maltolate greatly enhances the toxicity of Al both in vitro (Levesque et al., 2000Go; Ohyashiki et al., 2002Go; Tsubouchi et al., 2001Go) and in vivo (Savory et al., 1999Go). Al-malt was prepared as previously described in detail (Bertholf et al., 1989Go), and a 20 mM stock solution was prepared immediately prior to use. Al-malt forms a metastable solution with a pH of 7. Each molecule of Al binds three molecules of maltolate, and for this reason the maltolate control was added to cells at 3x molar concentrations. The role of de novo protein synthesis in the observed toxicity was examined using cell cultures pretreated with CHX (0.5 µM final concentration) for 30 min prior to treatment with Al-malt or maltolate. All solutions were filter sterilized using 0.22-µm syringe filters immediately after preparation.

Determination of Al cytotoxicity in N2a cells. Mitochondrial enzyme activity, an indirect measure of the number of viable respiring cells, was determined using the 3[4,5-dimethyl thiazolyl-2]2,5-diphenyl tetrazolium bromide (MTT) assay. N2a cells were seeded in 96-well culture plates at a density of 20,000 cells/well in 100 µl volume and allowed to grow for 18 h before treatments. Cells were treated by adding 100 µl of a 2x concentration of the respective reagent (AlCl3, Al-malt, or maltolate as detailed in figures), and 24 h later, MTT was added to each well at a final concentration of 0.5 mg/ml for 4 h. Media was removed and the purple formazan crystals were dissolved in 100% dimethyl sulfoxide. Absorbance at 570 nm was determined using a PowerWaveXTM absorbance microplate reader (Bio-Tek Instruments, Inc., Winooski, VT). Data are expressed as relative absorbance at 570 nm.

Cytotoxicity was determined using the lactate dehydrogenase (LDH) release assay as previously described (He et al., 2002Go). N2a cells were seeded as described above for the MTT assay. Treatments were added after 18 h as 2x concentrations. Plates were then incubated for 24 h, following which 40 µl of the supernatant was added to a new 96-well plate to determine LDH release, and 40 µl of 6% Triton X-100 was added to the original plate for determination of total LDH. An aliquot of 100 µl of 4.6 mM pyruvic acid in 0.1 M potassium phosphate buffer (pH 7.5) was mixed with the supernatant using repeated pipetting. Then 100 µl of 0.4 mg/ml reduced ß-NADH in 0.1 M potassium phosphate buffer (pH 7.5) was added to the wells, and the kinetic change in absorbance at 340 nm was read for 1 min using a PowerWaveXTM absorbance microplate reader. This procedure was repeated with 40 µl of the total cell lysate to determine total LDH U/well. A change of 0.001 absorbance units/min is equivalent to 1 U/l of LDH activity (Jemmerson et al., 2002Go). The percentage of LDH release was determined by dividing the LDH released into the media by the total LDH following cell lysis in the same well.

Determination of DNA synthesis as an index of cell proliferation. The effects of Al treatment on N2a cell proliferation and DNA synthesis was determined using a modification of the [methyl-3H]thymidine incorporation assay previously described (Johnson and Sharma, 2001Go). N2a cells were treated with Al-malt for 24 h, following which 25 µCi/ml of [methyl-3H]thymidine was added for an additional 6 h. Cells were then harvested onto glass fiber filter paper (Cambridge Technology, Watertown, MA) using a cell harvester (PHD, Cambridge Technology). The harvested cells were lysed with deionized water, and filters dried with 95% ethanol. The filter papers were placed in scintillation vials containing 2 ml of liquid scintillation cocktail (Ready-Solv, Beckman, Fullerton, CA) and counted in a liquid scintillation counter (Pharmacia, Turku, Finland). Proliferative responses (uptake of 3H-thymidine) were expressed as stimulation index [disintegrations per minute (DPM)treated/DPMcontrol].

Determination of apoptosis using the acridine orange/ethidium bromide assay. The ability of acridine orange and ethidium bromide dual-staining to differentiate live from apoptotic and necrotic cells has been useful in cell death research. The assay was performed as previously described (Kern and Kehrer, 2002Go). N2a cells were treated with Al-malt for 24 h, following which the cells were harvested by trypsinization. The floating and adherent cells were collected and combined. Cells (106) were centrifuged at 200 x g for 10 min at 4°C and resuspended in 1 ml PBS, and 2 µl of acridine orange and 2 µl of ethidium bromide (final concentration 200 ng/ml for each) were added to each tube, and the cells were stained for 5 min. Two-parameter fluorescence was acquired from 20,000 individual cells per sample using an EPICS XL/MCL flow cytometer (Coulter Cytometry, Hialeah, FL, USA) with a 488 nm argon-ion laser. Green emissions from acridine orange and red emissions from ethidium bromide were captured at 525 and 620 nm, respectively. Live, apoptotic, and necrotic populations were analyzed using WinMDITM flow analysis software. Discrimination between cell populations was based on previously published diagrams and descriptions (Kern and Kehrer, 2002Go; Liegler et al., 1995Go).

Determination of phosphatidyl serine externalization: annexin V binding assay. The externalization of phosphatidyl serine is an early event in apoptosis and serves as a signal for phagocytosis by macrophages (Anderson et al., 2002Go). Flow cytometry analysis was performed to determine this event using the annexin V binding assay (BD Biosciences Pharmingen, San Diego, CA). N2a cells were treated with Al-malt for 24 h, following which 100,000 cells were washed 2x in cold PBS and resuspended in 100 µl 1x binding buffer. Five µl of annexin V-FITC and 5 µl of PI (final concentrations 2.5 µg/ml) were added and gently mixed, followed by incubation protected from light for 15 min. Cells were acquired within 60 min using an EPICS XL/MCL flow cytometer. The fluorochrome was excited using the 488 nm line of argon-ion laser, and annexin V and PI emissions were monitored at 525 and 620 nm, respectively. A total of 20,000 events were acquired for each sample. The following controls were run at the time of assay (1) unstained cells, (2) annexin V only, and (3) PI only.

Determination of caspase 3 activation. Caspase 3-like activity was determined using the CaspACETM fluorometric activity assay (Promega Corporation, Madison, WI) with modifications as noted. Recently it was reported that approximately 15% and 58% of the active caspase 3 enzyme is released into the media within 4 and 24 h, respectively, following induction of apoptosis (Jemmerson et al., 2002Go). Traditional assays require a wash step prior to lysis of the cells that may result in the loss of the active enzyme and a false negative result. For this reason, we lysed the cells in the treatment media using 1% Triton X-100. N2a cells were treated in 24-well plates at a density of 200,000/ml for 24 h, following which Triton X-100 was added to a final concentration of 1%. The cells were lysed by trituration and centrifuged at 10,000 x g for 10 min to remove cell debris. The supernatant was assayed for active caspase 3-like activity using the CaspACETM system according to manufacturer's instructions. The fluorescence of 7-amino-4-methyl coumarin (AMC) following cleavage of the substrate was determined using a Spectramax Gemini fluorescent plate reader (Molecular Devices, Irvine, CA, USA). The fluorescence signal was digitized and analyzed using SoftMax ProTM (version 3.1.1, Molecular Devices), and the concentration of caspase 3-like activity determined from an AMC standard curve.

Cell cycle analysis. The cell cycle distribution of N2a cells treated with Al-malt was determined using flow cytometry. Cells were treated with Al-malt for 24 h and then floating and trypsinzed cells were resuspended in nuclear isolation media (NIM; 50 µg/ml PI, 1 mg/ml RNase A, 0.1% Triton X-100). Nuclear DNA content from 50,000 cells was determined using an EPICS XL/MCL flow cytometer with a 488 nm argon-ion laser and an emission wavelength of 620 nm. DNA histograms were analyzed using WinMDITM flow analysis software.

Analysis of cellular and nuclear morphology. Cellular morphology was examined using phase and scanning electron microscopy, and nuclear morphology was examined using an epifluorescence microscope following staining with Hoechst 33258 (H33258). Cells were seeded (2 x 105/ml) in 6-well plates for phase contrast and nuclear morphology imaging. Following 24-h treatment with Al-malt, cells were stained with H33258 (1 µg/ml) for 5 min, and phase and fluorescence microscopy was performed using an IX71 inverted microscope (Olympus America, Inc., Melville, NY). Digital images were captured using a MagnaFire SP® digital camera.

Scanning electron microscopy was performed on cells seeded on 13-mm ThermanoxTM cover slips (Nalge Nuc International, Rochester, NY) in 24-well plates and treated with Al-malt for 24 h. Following the treatment, cover slips were removed from the well and rinsed with ice cold PBS for 5 s and immediately plunge-frozen in liquid nitrogen. The frozen monolayers were freeze-dried for 18 h at –50°C and carbon coated using a Denton DV502A vacuum evaporator (Denton Vacuum, Moorsetown, NJ). This fixation method does not affect cellular morphology (Fernandez-Segura et al., 1999Go). Imaging was conducted using a LEO 982 field emission scanning electron microscope (Leo Electron Microscopy Inc., Thornwood, NY).

Real-time PCR analysis of gene expression. N2a cells were treated with Al-malt for 24 h, followed by preservation of RNA with RNAlater (Qiagen, Valencia, CA) according to manufacturer's directions. Total cellular RNA was isolated using RNeasy kits (Qiagen) according to the instructions provided. One microgram of RNA was reverse-transcribed using oligo dT and 60 U of Superscript II (Life Technologies, Grand Island, NY). Real-time Sybr Green primers (see Table 1) for Bcl-2, Bax, Mdm2, p53 and GAPDH (reference gene) were designed using Beacon Designer (PREMIER, Biosoft International, Palo Alto, CA). Template was combined with primers and 2X Taqman® Universal Sybr Green PCR Master Mix (contains PCR buffer, passive reference dye ROX, deoxynucleotides, uridine, uracil-N-glycosylase, and AmpliTaq Gold® DNA polymerase; Perkin-Elmer, Applied Biosystems, Foster City, CA) in a 25-µl reaction volume, and amplification was performed using an ABI Prism® 7900HT (PE, Applied Biosystems) real-time thermal cycler. Cycling conditions were 2 min at 50°C; 10 min at 95°C; 50 cycles of 92°C for 30 s; 60°C for 1 min. Real-time fluorescence detection was performed during the 60°C annealing/extension step of each cycle. Melt-curve analysis was performed on each primer set to ensure that no primer dimmer or nonspecific amplification was present under the optimized cycling conditions. The fold difference in mRNA expression between treatment groups was determined using the relative quantification method utilizing real-time PCR efficiencies (Pfaffl et al., 2002Go) and normalized to the housekeeping gene, GAPDH, thus comparing relative CT changes between control and experimental samples. Prior to conducting statistical analyses, the fold change from the mean of the control group was calculated for each individual sample (including individual control samples to assess variability in this group).


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TABLE 1 Sybr Green Primer Sets Designed Using Beacon Designer

 
Statistical analysis. All statistical analyses were performed using Minitab statistical software (Minitab Inc., State College, PA). Treatment effects were determined using one-way analysis of variance followed by Tukey's post hoc analysis. A value of p < 0.05 was considered significant unless indicated otherwise.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Al-Malt Induces Cell Death in Neuro-2a Cells
The complex speciation chemistry of Al presents a difficulty when comparing the cytotoxicity of various forms used for in vitro experiments. Our first goal was to determine the susceptibility of N2a cells to an inorganic and an organic form of Al. Treatment of N2a cells for 24 h with up to 1000 µM of AlCl3 did not induce cell death as indicated by the MTT cytotoxicity assay (Fig. 1). In contrast, a dose-dependent increase in cell death was observed in N2a cells treated with Al-malt. Mitochondrial function was reduced to 80 and 40% of control levels in cells treated with 250 and 500 µM Al-malt, respectively, for 24 h (Fig. 1). Previously, it was reported that maltolate can cause cell death in N2a cells (Hironishi et al., 1996Go). Therefore, we tested the ability of maltolate to induce cell death in our system. Since three molecules of maltolate are bound to each ion of Al, we used 3x molar concentration of maltolate as an appropriate control group. Contrary to the results of Hironishi et al. (1996)Go we did not observe any change in viable cell number by the MTT assay (Fig. 1). It can be concluded that 24-h exposure is insufficient for the manifestation of maltolate toxicity in N2a cells. N2a cells exposed to 1500 µM maltolate did show a small decrease in viability, although the effect was not significant. Perhaps toxicity would be evident at 72 h, as seen previously in this cell line (Hironishi et al., 1996Go) and in brain rotation aggregate cultures (Johnson and Sharma, 2003Go). The findings of the mitochondrial MTT assay were confirmed by examining the release of lactate dehydrogenase (LDH) from the cells. Figure 1 indicates that Al-malt induced a dose-dependent increase in the release of LDH, indicating a loss of integrity of the cell membrane. The increase was significant at concentrations above 62.5 µM. Maltolate alone did not increase LDH release from N2a cells (data not shown).



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FIG. 1. The effects of two different aluminum compounds on the viability of N2a neuroblastoma cells. Cells were treated with AlCl3 (1–1000 µM), Al-malt (62.5–500 µM), and maltolate (1500 µM) at the indicated concentrations for 24 h. The relative number of live cells was then determined using the MTT assay. The dotted line represents the LDH release (% of total) from cells treated with Al-malt (62.5–500 µM). Mean ± SE (n = 5 wells/treatment). Data are representative of three independent experiments with similar results. Significantly different from the control group at *p < 0.01, **p < 0.001.

 
The toxicity of Al-malt was also evident in the marked drop in DNA synthesis by 24 h of treatment (Fig. 2). Incorporation of 3H-thymidine into N2a was completely abolished by 500 µM Al-malt. Maltolate alone did not decrease DNA synthesis at the doses tested. All concentrations of maltolate except for 1500 µM induced DNA synthesis in N2a cells, indicative of a proliferative effect (Fig. 2). These findings confirm that the enhanced toxicity of Al-malt is specific for the complex and not simply due to the presence of maltolate.



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FIG. 2. Treatment of N2a cells with Al-malt reduced DNA synthesis, an index of proliferation. Cells were treated with Al-malt (62.5–500 µM) and maltolate (1500 µM) at the indicated concentrations for 24 h. 3H-Thymidine was then added for an additional 4 h, and DNA synthesis was determined according to the Materials and Methods. Mean ± SE (n = 5 wells/treatment). Data are representative of three independent experiments with similar results. Significantly different from the control group at *p < 0.05, **p < 0.001.

 
Apoptosis and Necrosis Are Involved in Al-Malt Neurotoxicity
Dual staining with cell-permeant acridine orange and cell-impermeant ethidium bromide is an effective flow cytometric method for discriminating apoptotic from necrotic cell death (Lecoeur, 2002Go). Live cells (Fig. 3, population L) stain with acridine orange and fluoresce green with low orange-red signal from ethidium bromide. Apoptotic cells (Fig. 3, population Ap) exhibit a reduced green fluorescence but maintain a low ethidium bromide signal. The reduction in acridine orange signal is likely due to nuclear fragmentation and cell shrinkage. Necrotic cells (Fig. 3, population N) lose the ability to exclude ethidium bromide and, as a result, fluoresce as bright orange-red (Fig. 3). Treatment of N2a cells with Al-malt resulted in a dose-dependent increase in the apoptotic and the necrotic populations (Figs. 3c–3f). The group treated with maltolate alone (Fig. 3b) showed a slight increase in the apoptotic population over that of the control (Fig. 3a). This may reflect the beginning of an increase in apoptosis as reported earlier (Hironishi et al., 1996Go) or may represent a slowing of the normal apoptotic turnover of cells observed in all cell cultures. These cell changes in response to Al-malt treatment, nuclear condensation, and cell shrinkage are early events in the apoptotic cascade.



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FIG. 3. Al-malt induces apoptosis and necrosis in N2a cells. Cells were treated with Al-malt and maltolate at the indicated concentrations for 24 h. Discrimination of live, apoptotic, and necrotic cell populations was determined using the acridine orange/ethidium bromide flow cytometry assay as outlined in the Materials and Methods. Live cells are defined by the oval labeled L, and apoptotic and necrotic populations are labeled Ap and N, respectively. (a) control; (b) 1500 µM maltolate; (c) 62.5 µM Al-malt; (d) 125 µM Al-malt; (e) 250 µM Al-malt; (f) 500 µM Al-malt. Data are representative of three independent experiments with similar results.

 
The externalization of phosphatidyl serine (PS) is another early event in apoptosis and represents a flag to induce phagocytosis by mononuclear cells (Anderson et al., 2002Go). This event was determined using the annexin V binding assay and flow cytometry. Al-malt concentration-dependently increased the externalization of PS as shown in Figures 4c–4f. A slight increase in annexin V binding was also observed in the maltolate-treated cultures (Fig. 4b), confirming the findings of the acridine orange/ethidium bromide assay. Annexin V can also bind to PS on the inner leaflet when the integrity of the plasma membrane is compromised and is indicative of necrosis or secondary necrosis following apoptosis. To control for this, PI, a cell-impermeant nuclear stain was used. Live cells are displayed in the lower left quadrant and are negative for both stains. The upper right quadrant of the panels in Figure 4 represents cells that are staining for annexin V and PI and have thus undergone necrotic cell death. The cells in the lower right quadrant are the early apoptotic cells showing a characteristic redistribution of PS. The amount of cells in the upper and lower right quadrants were increased by Al-malt. Thus, the annexin V binding assay confirms the coexistence of apoptotic and necrotic cell death in N2a cells treated with Al-malt.



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FIG. 4. Externalization of phosphatidyl serine is induced in cells treated with Al-malt. Cells were treated with Al-malt and maltolate at the indicated concentrations for 24 h, following which annexin V binding was performed according to the Materials and Methods. Live cells are in the lower left quadrant. The upper right quadrant represents necrotic and late apoptotic cells, and the lower right quadrant represents early apoptotic cells. (a) control; (b) 1500 µM maltolate; (c) 62.5 µM Al-malt; (d) 125 µM Al-malt; (e) 250 µM Al-malt; (f) 500 µM Al-malt. Data are representative of three independent experiments with similar results.

 
Microscopic analysis of cellular and nuclear morphology also suggests that apoptosis and necrosis are both induced in N2a cells following treatment with Al-malt. Cells treated with 500 µM Al-malt show cellular shrinkage (Fig. 5c) as well as nuclear condensation and fragmentation (Fig. 5d inset, arrow), both hallmark features of apoptosis. In addition, treated cells exhibit dramatic membrane blebbing (Fig. 5j, arrow). Signs of necrosis are also evident in the same cultures that display apoptosis. Cell swelling (Fig. 5c) and loss of membrane integrity without nuclear condensation (Fig. 5d inset, arrow head) are evident in cultures treated with 500 µM Al-malt. The loss of membrane integrity is also apparent in the scanning electron micrograph of cells treated with Al-malt (Fig. 5j, arrow head). All of these features of cell death were not evident in control cultures (Figs. 5a, 5b, and 5i). Pretreatment with CHX (CHX controls are shown in Figs. 5e and 5f) greatly reduced the cell shrinkage (Fig. 5g) and nuclear alterations (Fig. 5h) induced by Al-malt. In addition, neuronal processes were still evident in cultures pretreated with CHX (Fig. 5g, arrows), whereas treatment with Al-malt alone completely eliminated process formation (Fig. 5c).



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FIG. 5. Cellular and nuclear morphology indicative of apoptosis and necrosis can be seen in N2a cells treated with Al-malt. Cells were treated with Al-malt and maltolate at the indicated concentrations for 24 h and then examined by light microscopy (all x200) and scanning electron microscopy (SEM). CHX was added to some cell cultures 30 min before the addition of Al-malt. (a) control phase contrast; (b) control H33258; (c) 500 µM Al-malt phase contrast; (d) 500 µM Al-malt H33258; (e) CHX phase contrast; (f) CHX H33258; (g) 500 µM Al-malt + CHX phase contrast; (h) 500 µM Al-malt + CHX H33258; (i) control SEM; (j) 500 µM Al-malt SEM. Micrographs are representative of two independent experiments with similar results.

 
Caspase 3 is activated in N2a cells treated with Al-Malt. The activation of caspase 3 is an integral step in the majority of apoptotic events. This enzyme belongs to the cysteine protease family and is responsible for cleaving substrates, such as DNA fragmentation factor that can go on to damage DNA (Sharif-Askari et al., 2001Go), and is thus considered to be an effector caspase. Caspase 3 activity was markedly increased in a concentration-dependent manner in N2a cells treated with Al-malt (Fig. 6). A slight increase in caspase 3 activity was noted in the maltolate-treated cells (open bar in Fig. 6) indicative of a small increase in apoptosis. The specificity of the assay was demonstrated by using a specific inhibitor of caspase 3, Ac-DEVD-CHO, which abolished the activity in Al-malt-treated lysates (solid bar in Fig. 6). The caspase 3 activity of washed cells was much lower than that present in cells lysed in the treatment media (data not shown). This could be due to removal of apoptotic cells during washing, or more likely, due to the release of caspase 3 from dying cells into the media as reported previously (Jemmerson et al., 2002Go).



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FIG. 6. Treatment of N2a cells with Al-malt results in activation of caspase 3. Cells were treated with Al-malt (62.5–500 µM) and maltolate (1500 µM, open bar) at the indicated concentrations for 24 h, and then caspase 3-like activity was determined using the CaspACETM system as outlined in the Materials and Methods. To confirm the specificity of the assay, lysates from cells treated with 500 µM Al-malt were pretreated with Ac-DEVD-CHO (solid bar), a specific inhibitor of caspase 3. Mean ± SE (n = 3 wells/treatment). The experiment was repeated three times with similar results. Significantly different from the control group at *p < 0.001.

 
Al-Malt-Induced Apoptosis Requires de Novo Protein Synthesis
Apoptosis is an active form of cell death and can require de novo protein synthesis to initiate and/or complete the process (Chow et al., 1995Go). Cycloheximide is a potent inhibitor of gene translation and has been used extensively in apoptosis research. Pretreatment of N2a cells with 0.5 µg/ml of CHX for 30 min prior to the addition of Al-malt produced a marked inhibition of cell death as evidenced by reduced release of LDH into the media (Fig. 7, solid bars). Flow cytometric examination using acridine orange/ethidium bromide dual staining showed that CHX almost completely inhibited apoptosis but not necrosis (Fig. 8). These findings were confirmed by cell cycle analysis and the detection of hypodiploid nuclei. Al-malt dose-dependently increased the hypodiploid population, and this was abolished in cells pretreated with CHX (Fig. 8). Al-malt appeared to induce cell cycle arrest in the G2/M phase, a phenomenon often associated with toxicant-induced apoptosis in cultured cells (Zaffaroni et al., 2001Go). Therefore, perturbations of cell cycle progression may be involved in Al-malt-induced neurotoxicity.



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FIG. 7. Al-malt-induced cell death in N2a cells is inhibited by pretreatment with the protein synthesis inhibitor cycloheximide. Cells were treated with Al-malt (62.5–500 µM) for 24 h in the presence (solid bars) or absence (hatched bars) of a 30 min pretreatment with CHX (0.5 µg/ml). The release of LDH from the cells was then determined as outlined in the Materials and Methods. Mean ± SE (n = 5 wells/treatment). Data are representative of three independent experiments with similar results. Significantly different from the control group at *p < 0.05, **p < 0.001. §Significantly different from the respective group treated with Al-malt only at p < 0.005.

 


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FIG. 8. Al-malt-induced apoptosis is inhibited by cycloheximide. Cells were treated with Al-malt (62.5–500 µM) at the indicated concentrations for 24 h in the presence or absence of CHX (0.5 µg/ml; 30 min pretreatment prior to addition of Al-malt). Discrimination of live, apoptotic, and necrotic cell populations was determined using the acridine orange/ethidium bromide flow cytometry assay as outlined in the Materials and Methods. Live cells are defined by the oval labeled L, and apoptotic and necrotic populations are labeled Ap and N, respectively. The protection of N2a by pretreatment with CHX was confirmed by examining the cell cycle in response to treatment. CHX pretreatment inhibited the appearance of a hypodiploid DNA peak. Data are representative of three independent experiments with similar results.

 
Confirming a role for de novo protein synthesis in the initiation of apoptotic cell death induced by Al-malt is the finding that pretreatment of N2a cells with CHX prevented the activation of caspase 3 (Fig. 9). The activation of caspase 3 by Al-malt did not increase significantly beyond 250 µM (Figs. 6 and 9). Pretreatment with CHX completely abolished caspase 3 activation induced by 250 µM Al-malt but only reduced the activation in cells treated with 500 µM Al-malt (Fig. 9). The same trend is evident in Figure 7, showing complete inhibition of cell death by CHX in cells treated with 250 µM but only partial inhibition in cells treated with 500 µM Al-malt. It is possible that 0.5 µg/ml of CHX does not completely inhibit transcription in N2a cells, thus accounting for these results. Higher doses of CHX need to be investigated to confirm this hypothesis.



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FIG. 9. Inhibition of protein synthesis inhibits caspase 3 activation by Al-malt. Cells were treated with Al-malt (62.5–500 µM) at the indicated concentrations for 24 h in the presence or absence of CHX (0.5 µg/ml; 30 min pretreatment prior to addition of Al-malt), and then caspase 3-like activity was determined using the CaspACETM system as outlined in the Materials and Methods. Mean ± SE (n = 3 wells/treatment). The experiment was repeated three times with similar results. Significantly different from the control group at *p < 0.001.

 
Al-Malt Alters Expression of Key Regulators of the p53 Apoptotic Pathway
Previous studies have demonstrated the presence of p53 protein in N2a cells and that this pathway can induce apoptosis in these cells (Jin et al., 2002Go). Treatment of N2a cells with 250 µM Al-malt increased the expression of p53 nearly twofold (Fig. 10). A concomitant increase in Mdm2 expression was observed, suggesting the induction of a negative regulatory response to the increase in p53 expression. These changes were accompanied by a marked down-regulation of Bcl2 and an increased expression of BAX. Thus, Al-malt induced a marked shift in the Bcl2/BAX expression ratio toward a state associated with apoptosis.



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FIG. 10. Al-malt treatment up-regulates gene expression for pro-apoptotic factors. Cells were treated with 250 mM Al-malt for 24 h, and total RNA was immediately extracted and converted to cDNA. The expression of anti-apoptotic (Bcl2 and Mdm2) and pro-apoptotic (BAX and p53) genes was analyzed by real-time PCR using SYBR Green I. Target gene expression is normalized to GAPDH expression, and the results are expressed as fold change from media-only control cultures. Mean ± SE (n = 6 wells/treatment). Results are representative of three independent experiments showing similar changes. Significantly different from the control group at *p < 0.05; **p < 0.001.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis has been implicated as a prominent form of cell death in many human neurodegenerative diseases including AD (Marx, 2001Go, 2003Go). Understanding the role of environmental factors in the induction of apoptosis in the brain or alterations in natural apoptotic processes is vital to defining the etiology of neurodegenerative diseases. Recent studies have generated a strong body of evidence indicating the potential of Al-malt to induce apoptosis and neurodegeneration in the brain (Ghribi et al., 2001aGo, 2002aGo; Savory et al., 1999Go) as well as in vitro (Griffioen et al., 2004Go; Kawahara et al., 2003Go; Ohyashiki et al., 2002Go; Savory et al., 2001Go). In vitro models represent an effective system for determining the mechanisms involved in Al-malt-induced neurodegeneration. The present study focused on characterizing the mode of cell death in N2a cells treated with Al-malt. The results clearly show that apoptosis is induced by Al-malt and that necrosis also plays a role in the neurotoxicity. We also determined the effects of pharmacological intervention on the toxicity of Al-malt. Al-malt-induced apoptosis was dependent upon de novo protein synthesis, as evidenced by a marked inhibition of toxicity in the presence of CHX. Necrosis induced by Al-malt was not inhibited by CHX.

The manner in which a cell dies can have a great impact on the resulting response in the surrounding tissue. Death by necrosis has been shown to act as a natural adjuvant, inducing oxidative stress and the production of numerous pro-inflammatory cytokines (Anderson et al., 2002Go). This is due to the indiscriminant release of the cytoplasmic contents from the dying cells. The result is an area of inflammation leading to immune-mediated damage of innocent cells surrounding the initial insult. In contrast, death by apoptosis is a controlled event, usually with minimal loss of membrane integrity until the later stages termed secondary necrosis. Instead, the cytoplasmic contents are systematically degraded from within. This type of cell death often involves phagocytosis by resident tissue macrophages and the release of anti-inflammatory cytokines (Fadok et al., 1998Go). However, a recent study suggested that this contention may not always apply. Jemmerson et al. (2002)Go showed that cytochrome c, active caspase 3, and LDH were all released from apoptotic cells and that release began within 2 h of the initiating stimulus. Previously, it was thought that all of these proteins were degraded in the cytoplasm of apoptotic cells. Therefore, apoptotic cell death may also substantially contribute to death-induced secondary inflammation and damage to surrounding tissues. The findings of this study confirm that LDH and active caspase 3 are released from apoptotic cells, since inhibition of apoptosis with CHX reduced LDH release and caspase 3 activity in the media. Results presented here support the hypothesis that the apoptosis seen in vivo following treatment with Al-malt could contribute to secondary immune-mediated damage and further neurodegeneration.

Caspases are important mediators of apoptosis, and caspase activation has been demonstrated in Al-malt-induced neurodegeneration (Ghribi et al., 2001aGo, 2002aGo,bGo). Our in vitro N2a cell model also showed strong caspase 3 activation in response to Al-malt treatment. Inhibition of protein synthesis using CHX inhibited apoptotic cell death and the activation of caspase 3 in Al-malt-treated cells, implicating de novo protein synthesis in the activation of this death effector. Protein synthesis could affect the regulation of caspase 3 in two ways: (1) new pro-caspase 3 synthesis and (2) synthesis of other proteins required for caspase 3 activation. Our results show that the expression of BAX mRNA is increased in N2a cells following treatment with Al-malt. BAX is an important mediator of the mitochondrial intrinsic pathway leading to caspase 3 activation and apoptosis and likely represents one of the pro-apoptotic factors down-regulated by CHX in this system.

The involvement of de novo protein synthesis in toxicant-induced apoptosis has been demonstrated previously (Chow et al., 1995Go). Our finding of a marked decrease in the neurotoxicity of Al-malt in the presence of the protein synthesis inhibitor CHX suggests that Al-malt-induced apoptosis is at least partially dependent on de novo protein synthesis. Several candidate proteins have been identified from in vivo studies in the rabbit brain. The pro-apoptotic protein BAX has been implicated in Al-malt-induced neurodegeneration in the rabbit (Ghribi et al., 2001aGo, 2002aGo; Savory et al., 1999Go). Present data demonstrates that Al-malt increased the expression of BAX in N2a cells. Studies with other chemicals including nitric oxide (NO) showed that apoptosis can be accompanied by an increase in BAX protein synthesis (Chen et al., 2002Go). Inhibition of protein synthesis partially inhibited the apoptosis suggesting that de novo synthesis of BAX may be responsible for the apoptosis induced by NO. The demonstration of increased BAX immunoreactivity in the CA1 region of the hippocampus (Ghribi et al., 2002bGo; Savory et al., 1999Go) of rabbits treated with Al-malt and our data showing increase BAX mRNA expression strongly support a role for new BAX synthesis in the induction of apoptosis in these models. We also observed a dramatic down-regulation of Bcl2 expression following treatment of N2a cells with Al-malt. A similar down-regulation of Bcl2 immunoreactivity is observed in the brain of aged rabbits following treatment with Al-malt (Savory et al., 1999Go). It is clearly evident that the intrinsic pathway of apoptosis involving altered Bcl2/BAX ratio is critical to Al-malt apoptosis.

The tumor suppressor gene product p53 is an important activator of the intrinsic apoptotic pathway. Here, we demonstrate for the first time that Al-malt induces the expression of p53 in neuronal cells. Up-regulation of p53 is commonly observed following exposure to DNA-damaging agents such as UV light. Increased production of growth arrest and DNA damage inducible factor 153 suggests that DNA damage may be induced in the brain of Al-malt-treated rabbits (Ghribi et al., 2001bGo) and could lead to activation of p53. Tumor suppressor p53 has been shown to differentially regulate Bcl2 and BAX levels both in vitro and in vivo (Miyashita et al., 1994bGo). Increased production of BAX following p53 activation is due to p53 being a direct transactivator of BAX gene expression (Miyashita and Reed, 1995Go). Importantly, a p53-dependent negative response element is present in the 5' untranslated region of the Bcl2 gene and is responsible for potent transcriptional down-regulation following p53 activation (Miyashita et al., 1994aGo). Our observations of decreased Bcl2 and increased BAX expression concomitant with increased p53 expression suggest that apoptosis induced by Al-malt in N2a cells may be controlled by the p53 pathway, and further studies are warranted.

In conclusion, the results of the present study strongly suggest that apoptosis is a prominent form of cell death in N2a cells treated with Al-malt, thus supporting previous in vivo findings. However, necrotic cell death was also apparent in N2a cells and may play a role in the neurotoxicity of Al-malt observed in vivo. The substantial release of intracellular enzymes observed in these cells even when dying by apoptosis has implications for the involvement of secondary damage induced by immune activation and inflammation in Al-malt neurotoxicity. Furthermore, the inhibitory effect of CHX on Al-malt-induced cell death indicates that de novo protein synthesis is intimately involved in the resulting neurotoxicity. The observed shift in Bcl2/BAX ratio confirms in vivo findings, and our data suggest that p53 is a potential mediator of this effect. Detailed investigation of the p53 pathway in response to treatment with Al-malt will greatly improve our understanding of the mechanisms involved in Al neurotoxicity and may provide insight into the etiology of neurodegenerative diseases including AD. Such research may also lead to effective strategies for therapeutic intervention in human neurodegenerative diseases. The present in vitro model shows many of the molecular characteristics present in the brains of rabbits treated with Al-malt and thus is an important in vitro alternative for further mechanistic studies.


    ACKNOWLEDGMENTS
 
Partial support of this work by Center for Academic Excellence in Toxicology at the University of Georgia and Fred C. Davison Endowment is gratefully acknowledged.


    NOTES
 
1 Current address for V. J. Johnson, Toxicology abd Molecular Biology Branch, National Institute of Occupational Safety and Health, Morgan Town, WV 26505. Back

2 To whom correspondence should be addressed at Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602–7389. Fax: (706) 542-3015. E-mail: rpsharma{at}vet.uga.edu.


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