Mitochondrial Proteomic Analysis and Characterization of the

Mitochondrial Proteomic Analysis and Characterization of the Intracellular Mechanisms of Bis(7)-tacrine in Protecting against Glutamate-Induced Excitotoxicity in Primary Cultured Neurons Hongjun Fu,†,# Wenming Li,†,‡,# Yulin Liu,‡ Yuanzhi Lao,§ Wei Liu,‡ Cheng Chen,§ Hua Yu,† Nelson T. K. Lee,† Donald C. Chang,§ Peng Li,| Yuanping Pang,⊥ Karl W. K. Tsim,§ Mingtao Li,‡ and Yifan Han*,† Departments of Biochemistry and Biology, Hong Kong University of Science and Technology, Hong Kong, China, The Proteomics Laboratory, Zhongshan Medical College, Sun Yat-sen University, Guangzhou 510080, China, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China, and Mayo Foundation for Medical Education and Research, Rochester, Minnesota 55905 Received November 20, 2006

Increasing evidence supports that the mitochondrial dysfunction, mainly caused by abnormal changes in mitochondrial proteins, plays a pivotal role in glutamate-induced excitotoxicity, which is closely associated with the pathogenesis of acute and chronic neurodegenerative disorders, such as stroke and Alzheimer’s disease. In this study, post-treatment of cerebellar granule neurons with bis(7)-tacrine significantly reversed declines in mitochondrial membrane potential, ATP production, and neuronal cell death induced by glutamate. Moreover, this reversal was independent of NMDA antagonism, acetylcholinesterase inhibition, and cholinergic pathways. Using two-dimensional differential in-gel electrophoresis, we conducted a comparative analysis of mitochondrial protein patterns. In all, 29 proteins exhibiting significant differences in their abundances were identified in the glutamate-treated group when compared with the control. The expression patterns in 22 out of these proteins could be reversed by post-treatment with bis(7)-tacrine. Most of the differentially expressed proteins are involved in energy metabolism, oxidative stress, and apoptosis. In particular, the altered patterns of four of these proteins were further validated by Western blot analysis. Our findings suggest that multiple signaling pathways initiated by the altered mitochondrial proteins may mediate glutamate-induced excitotoxicity and also offer potentially useful intracellular targets for the neuroprotection provided by bis(7)-tacrine. Keywords: Bis(7)-tacrine • glutamate • mitochondria • cerebellar granule neurons • two-dimensional differential in-gel electrophoresis • MALDI-TOF/MS

Introduction Glutamate is a major excitatory neurotransmitter in mammalian central nervous systems (CNS) and plays an important role in a wide variety of CNS functions. Overstimulation of its postsynaptic receptors, mainly the NMDA subtype, causes glutamate excitotoxicity, which underlies neuronal loss in ischemic and traumatic brain injury1 and also likely contributes to dysfunction in chronic forms of neurodegeneration, such as Alzheimer’s disease (AD), Amyotrophic lateral sclerosis, Parkinson’s disease, and Huntington’s disease.2,3 It is well* To whom correspondence should be addressed. Tel: (852) 23587293, Fax: (852) 23581552, E-mail: [email protected]. † Department of Biochemistry, Hong Kong University of Science and Technology. ‡ Sun Yat-sen University. # Both authors contributed equally to this paper. § Department of Biology, Hong Kong University of Science and Technology. | Tsinghua University. ⊥ Mayo Foundation for Medical Education and Research. 10.1021/pr060615g CCC: $37.00

 2007 American Chemical Society

established that glutamate-induced neuronal death depends on entry of extracellular Ca2+ as a result of the activation of the NMDA subtype of glutamate receptors.4,5 Furthermore, increasing evidence suggests that mitochondrial dysfunction is a primary and early event in glutamate excitotoxicity.3,6 For example, the initial glutamate-triggered increase in cytoplasmic Ca2+ is associated with mitochondrial Ca2+ loading and slight mitochondrial depolarization followed by the opening of the permeability transition pore (PTP) and profound depolarization concurrent with a significant decline in cellular ATP and the loss of ionic homeostasis. Other early events may include the reduction of oxidative phosphorylation, the release of apoptogenic cytochrome c and the generation of reactive oxygen species and nitric oxide.7-9 However, the exact role of mitochondria in glutamate-induced neuronal cell death remains unclear. We reported that bis(7)-tacrine is a promising anti-AD agent based on its superior acetylcholinesterase (AChE) inhibition and memory enhancement.10,11 Interestingly, we also observed Journal of Proteome Research 2007, 6, 2435-2446

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research articles that bis(7)-tacrine engages in substantial neuroprotective activities in various types of neuronal injury. In brief, bis(7)tacrine can protect against ischemia-induced injury in primary cultured mouse astrocytes,12 hydrogen peroxide-induced apoptosis in pheochromocytoma cells,13 and amyloid beta proteinand glutamate-induced apoptosis in primary cultured neurons.14,15 We also found that, although it has a similar affinity and potency to memantine in blocking NMDA receptors, bis(7)-tacrine is much more potent than memantine in preventing glutamate-induced excitotoxicity in cerebellar granule neurons (CGNs),16 and our data (Figure 2B) has shown that bis(7)tacrine, but not memantine, added 1 h after glutamate challenge, can significantly reduce the cell death induced by glutamate in CGNs, indicating that this chemical may have some intracellular targets against glutamate excitotoxicity. Although bis(7)-tacrine inhibits neuronal nitric oxide synthase (NOS) as potently as does NG-monomethyl-L-arginine in vitro, post-treatment with bis(7)-tacrine is much more potent than post-treatment with NG-monomethyl-L-arginine in preventing glutamate-induced excitotoxicity,16 indicating that, besides neuronal NOS inhibition, bis(7)-tacrine may create other intracellular targets against glutamate-induced excitotoxicity. Furthermore, we have found that post-treatment with bis(7)tacrine, but not with memantine, can block declines in mitochondrial membrane potential and ATP production, two important indices for mitochondrial dysfunction, induced by glutamate (Figures 1A and 2B). Taken together, we hypothesized in this study that maintaining mitochondrial functions might be the intracellular mechanisms underlying bis(7)-tacrine against glutamate-induced excitotoxicity. Although the functional effects of glutamate on primary neuron cultures and isolated mitochondria and protective effects of bis(7)-tacrine on glutamate-induced excitotoxicity have been investigated,7,15,16 the relevant studies have evaluated only the changes in single proteins or single pathways, and they have not addressed the effects on the total cellular proteome or on the proteome of specific subcellular organelles, particularly that of the mitochondria. With the introduction of the study of proteomics, it has become possible to simultaneously analyze changes in multiple proteins. In the current study, to elucidate the intracellular mechanisms (especially the possible targets at mitochondria) of bis(7)-tacrine against glutamateinduced excitotoxicity, a comparative analysis of mitochondrial protein patterns was carried out in CGNs exposed to glutamate with or without bis(7)-tacrine added 1 h after the glutamate challenge using two-dimensional differential in-gel electrophoresis (2-D DIGE). In all, 29 proteins exhibiting significant differences in their abundances were identified in the glutamatetreated group when compared with the control. Of these, the changes in 22 could be reversed by bis(7)-tacrine. We found that most of the differentially expressed proteins are involved in energy metabolism, oxidative stress, and apoptosis.

Materials and Methods Materials and Apparatuses. The ATP bioluminescent somatic cell assay kit was purchased from Sigma. Tetramethylrhodamine ethyl ester (TMRE) was purchased from Molecular Probes. The sequence-grade modified trypsin was purchased from Promega. 2-D Quant kit, ECL Western blotting detection kit, Cyanine dyes (CyDyes: Cy2, Cy3, and Cy5), the immobilized pH 3-10 NL IPG strips, Deep Purple Total Protein Stain, and all other chemicals related to 2-D DIGE were purchased from Amersham Biosciences. Primary anti-voltage-dependent anion 2436

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channel 1 (VDAC1), -14-3-3β, -Raf-1 antibodies, and secondary anti-rabbit, -mouse, and -goat antibodies were purchased from Santa Cruz. Anti-cytochrome c oxidase subunit IV (COXIV) and anti-Histone H2A antibodies were purchased from Abcam. Anti-prohibitin was purchased from Calbiochem. Anti-heat shock protein 1, alpha (Hsp90) antibodies were purchased from Stressgen Bioreagents. PVDF membranes were purchased from Bio-Rad. Protease inhibitor cocktail was purchased from Roche. All other reagents were purchased from Sigma. All the devices and corresponding software used in the proteomic experiments were purchased from Amersham Biosciences, unless otherwise noted. Primary CGNs Cultures. Rat CGNs were prepared from 8-day-old Sprague-Dawley rats (the Animal Care Facility, the Hong Kong University of Science and Technology) as described previously.15 Briefly, neurons were seeded at a density of 2.0 × 106 cells/mL in basal modified Eagle’s (BME) medium containing 10% fetal bovine serum, 25 mM KCl, 2 mM glutamine, penicillin (100 U/mL), and streptomycin (100 µg/mL). Cytosine arabinoside (10 µM) was added to the culture medium 24 h after plating to limit the growth of non-neuronal cells. With the use of this protocol, 95-99% of the cultured cells were granule neurons. Experiments were performed 8 days after the cultures. Determination of Mitochondrial Membrane Potential (∆ψm). A confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) was used to monitor the changes in ∆ψm using the potential-sensitive dye, TMRE, as described by Dedkova and Blatter.17 CGNs were exposed to 0.2 µM TMRE for 15 min at 37 °C before the experiments. All the solutions contained 0.2 µM TMRE during the recordings. TMRE fluorescence was excited at 543 nm and recorded at 570-700 nm. For measurements of the time-dependent TMRE fluorescence changes, data were acquired every 20 s. Results were obtained by evaluating the fluorescence intensity (F) from selected areas within a cell, subtracting the background fluorescence, and dividing by the fluorescence intensity of the first image (F0), which was also determined by subtracting the background fluorescence. The results were expressed as F/F0. Measurement of ATP Production. The ATP levels of dissociated neurons under different treatments were measured with the bioluminescent somatic cell assay kit. The bioluminescent method utilizes an enzyme, luciferase, which catalyzes the formation of light from ATP and luciferin. The emitted light is linearly related to the ATP concentration and is measured in 1 min using a luminometer. Measurement of Cell Viability. The percentage of surviving neurons was estimated by determining the activity of mitochondrial dehydrogenases with 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay as described previously.15 Isolation of Mitochondria from CGNs Cultures. All the isolation steps were performed at 4 °C. Treated CGNs were washed 2 times in ice-cold PBS and harvested by centrifugation at 1000g for 10 min. The cell pellet was resuspended in 2 mL of homogenization buffer (0.5 M sucrose, 5 mM MgCl2, and 1 mM EGTA at pH 7.5 and 37.5 mM Tris-maleic acid at pH 6.4) containing 1% protease inhibitor cocktail and homogenized by gentle douncing. The homogenate was spun at 700g for 10 min to pellet the nuclei and the unbroken cells. The crude mitochondria were pelleted by spinning the supernatant at 9000g for 30 min, and soluble cytosolic proteins were collected by further centrifugation of the cytosolic fraction at 90 000g for

Proteomic Analysis of Bis(7)-tacrine against Glutamate

2 h. Then, the crude mitochondria were purified as described by Vance.18 Briefly, the crude mitochondria were suspended by hand homogenization in 1 mL of isolation buffer (250 mM mannitol, 5 mM HEPES, pH 7.4, 0.5 mM EGTA, and 0.1% bovine serum albumin), and the suspension was layered on top of 3 mL of buffer containing 225 mM mannitol, 5 mM HEPES, pH 7.4, 1 mM EGTA, 0.1% bovine serum albumin, and 30% (v/v) Percoll, before centrifugation at 95 000g for 30 min. A dense band containing purified mitochondria approximately 1/3 of the tube length down from the top of the polycarbonate ultracentrifuge tube was collected, diluted with the isolation buffer without 0.1% bovine serum albumin, and then washed twice by centrifugation at 6500g for 10 min to remove the Percoll. Finally, the Percoll-purified mitochondria pellet was dissolved in a standard cell lysis buffer (pH 8.5) containing 30 mM Tris, 2 M thiourea, 7 M urea, 4% (w/v) CHAPS, and 1% protease inhibitor cocktail. After sonication and centrifugation at 16 000g for 20 min, the supernatant protein concentration was evaluated with the 2-D Quant kit, and its concentration was adjusted to 5 mg/mL using the lysis buffer. Verification of Mitochondrial Purity. To verify the enrichment of the mitochondrial fractions, 30 µg of proteins from enriched mitochondrial, nuclear, and cytosolic fractions were separated on 12.5% SDS-PAGE gels and transferred to PVDF membranes. The blots were blocked by 5% non-fat milk in TBST for 1 h at room temperature and incubated overnight at 4 °C with mouse anti-COXIV, a marker specific for mitochondria; rabbit anti-Histone H2A, a specific marker for nuclei; or mouse anti-Raf-1, a cytosolic marker. The blots were then incubated with horseradish peroxidase labeled anti-mouse or rabbit IgG for 1 h at room temperature and then developed with an enhanced chemiluminescence method. Labeling of Mitochondrial Proteins with CyDyes. Mitochondrial protein samples (50 µg) were labeled separately with 0.4 nmol of CyDye (Cy3, Cy5), vortexed, and incubated for 30 min in the dark. A mixed sample composed of equal amounts of mitochondrial proteins from the three different groups was also labeled with Cy2. After 30 min, the labeling was stopped with 10 mM lysine. To control the labeling efficiency, labeled proteins (0.5 µg) were subjected to SDS-PAGE analysis, and the gels were scanned with the Typhoon 9400 scanner at the wavelengths corresponding to each CyDye, namely, 480 nm (Cy2), 532 nm (Cy3), and 633 nm (Cy5). Two-Dimensional In-Gel Electrophoresis. A 50-µg portion of each Cy3-, Cy5-, and Cy2-labeled sample was combined, and the sample volume was set to 450 µL with rehydration buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 10% (v/v) isopropanol, 5% (v/v) glycerol, 25 mM DTT, 0.002% (w/v) bromophenol blue, and 1% (v/v) pH 3-10 NL IPG buffer). The mixed sample from the above three CyDyes-labeled proteins was subjected to isoelectric focusing (24-cm IPG strips, pH 310 NL) on an IPGphor II isoelectric focusing unit. The strips were rehydrated and focused under an IPG strip cover fluid at 30 V for 12 h, 500 V for 1 h, 1 kV for 1 h, and 8 kV for 8-12 h until 85 000 Vh at 20 °C and a maximum current setting of 50 µA per strip. Prior to SDS-PAGE, each strip was equilibrated as described previously.19 The strips were loaded and run on 12.5% acrylamide gels at 20 °C in an Ettan Dalt six system at 2 W per gel for the first 50 min, followed by 17 W per gel until the bromophenol blue migrated to near the bottom of the gel. For protein identification and peptide sequencing, a preparative gel including 500 µg of the mixed sample (166.67 µg each) was performed in parallel.

research articles Gel Scanning and Image Analysis. Analytical gels were scanned directly between low-fluorescence glass plates with the Typhoon 9400 scanner at the three wavelengths specific to the CyDyes. The resolution was of 100 µm. The determination of the protein spot abundance was performed using the DeCyder 2-D Differential Analysis Software, v6.0. In brief, the three CyDye-labeled forms of each spot were co-detected within each gel. Ratios between samples and internal standard abundances were calculated for each protein spot with the DIA (Differential In-gel Analysis) module. Inter-gel variability was corrected by matching and normalization of the internal standard spot maps with the BVA (Biological Variance Analysis) module of the DeCyder software. Protein spots that had a statistically significant (p < 0.01) Student’s t test result for an increased or decreased expression level were accepted as being differentially expressed between the extracts under comparison. The preparative gel was post-stained with Deep Purple and also scanned with the Typhoon 9400 scanner at an excitation wavelength of 532 nm. The generated image was then matched to the analytical set. The protein positions of the significant differences were transferred to the Deep Purple-stained gel, and a pick list was created using the above DeCyder software. This pick list, along with the Deep Purple-stained gel, was transferred to the Ettan Spot Handling Workstation for automatic spot excision, digestion, extraction of the tryptic peptides, mixing, and spotting of digested proteins and matrix onto the MALDI-TOF target slides. MALDI-TOF Mass Spectrometry and Protein Identification. Tryptic peptide mass spectra were obtained using an Ettan MALDI-TOF Pro mass spectrometer. The instrument setting was reflector mode with 175 ns delay extraction time, 60-65% grid voltage, and 20 kV accelerating voltage. Laser shots at 250 per spectrum were used to acquire the spectra with mass ranges from 600 to 3000 Da. The trypsin autolytic fragment peaks (m/z 842.509 and 2211.104) served as internal standards for mass calibration. Protein identification was performed by searching the NCBInr protein database using MASCOT (http://www.matrixscience.com). Database searches were conducted allowing up to one missed trypsin cleavage and using the assumptions that the peptides were monoisotopic, oxidized at methionine residues, and carbamidomethylated at cysteine residues. Mass tolerance of 100 ppm was the window of error allowed for matching the peptide mass values. Probability-based MOWSE scores at greater than 57 were considered to be significant (p < 0.05). All protein identifications were in the expected MW and pI range based on position in the gel. Species search was limited to Rattus norvegicus. Western Blot Analysis. Thirty micrograms of mitochondrial and cytosolic proteins from each group were separated by 12.5% SDS-PAGE gel and transferred to PVDF membranes. The blots were blocked by 5% non-fat milk in TBST for 1 h at room temperature and incubated overnight at 4 °C with mouse antiCOXIV, mouse anti-prohibitin, goat anti-VDAC1, rabbit anti14-3-3β, or mouse anti-Hsp90. The blots were then incubated with horseradish peroxidase labeled anti-mouse, -goat, or -rabbit IgG for 1 h at room temperature and developed with an enhanced chemiluminescence method. Statistical Analysis. Analysis of variance followed by the SNK test was used for tests of significance between three or more groups. The comparison between two groups was performed by the Student’s t test. Results were expressed as means ( SEM, and statistical significance was accepted at p < 0.05. Journal of Proteome Research • Vol. 6, No. 7, 2007 2437

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Figure 1. Bis(7)-tacrine blocks the decline of mitochondrial membrane potential (∆ψm) induced by glutamate in CGNs. At 8 days in vitro (DIV), CGNs were scanned at the resting fluorescence intensity for 2 min and exposed to 75 µM glutamate for another 8 min, and then post-treated with 1 µM bis(7)-tacrine, 10 µM cyclosporine A, 5 µM memantine, 50 µM E2020, or 50 µM tacrine for 20 min. The time course curves were based on the relative fluorescence intensity of TMRE in each group (n ) 10 cells). The data, expressed as the percentage of F/F0, are the means ( SEM of three independent experiments (n ) 30 cells).

Results and Discussion Neuroprotection against Glutamate-Induced Mitochondrial Dysfunction and Cell Death by Bis(7)-tacrine. To test the effects of post-treatment with bis(7)-tacrine on the mitochondrial dysfunction induced by glutamate, we first established a model of glutamate-induced mitochondrial dysfunction. In our system, ∆ψm declined significantly after 4-min of exposure to 75 µM glutamate and then dropped to the lowest point after 15 min of exposure (Figure 1A); and glutamate (25-100 µM) also decreased the ATP level in CGNs in a concentrationdependent manner (Figure 2A). Calcium accumulation in the mitochondria and a subsequent opening of the permeability transition pore are involved in the decline of ∆ψm, the ATP decrease, the release of mitochondrial proteins, the translocation of some proteins into the mitochondria, and, finally, cell death.6,7 In this study, cyclosporin A (10 µM), which inhibits the opening of the mitochondrial permeability transition pore (PTP) by interaction with mitochondrial cyclophilin, prevented 2438


Figure 2. Bis(7)-tacrine reverses the decreases in ATP levels and cell viability induced by glutamate in CGNs. (A) At 8 DIV, CGNs were exposed to different concentrations of glutamate for 3 h and the ATP levels were measured. (B and C) At DIV8, CGNs were exposed to 75 µM glutamate, and different concentrations of bis(7)-tacrine, 10 µM cyclosporin A (CsA), 5 µM memantine (Mem), or 10 and 50 µM tacrine/E2020 were added 1 h later. After a 3 h glutamate challenge, the ATP levels were measured with an ATP assay kit. Cell survival was detected with an MTT assay after the CGNs were exposed to glutamate for 24 h. All the data, expressed as percentages of the control, are the means ( SEM of three separate experiments (n ) 6 wells for each group). *, p < 0.05; **, p < 0.01 versus control in A or versus glutamate alone group in B (ANOVA and SNK test).


the decline of ∆ψm (Figure 1A), the decrease of ATP levels (Figure 2B), and neuronal death (Figure 2B) induced by 75 µM glutamate in CGNs. These results are consistent with the findings from other groups6,20,21 and further confirm that mitochondrial dysfunction is a primary and early event in glutamate excitotoxicity. We next investigated the possible role of mitochondria in the neuroprotection of bis(7)-tacrine against glutamate excitotoxicity. Under the same experimental conditions, unlike memantine (an antagonist of NMDA receptors), post-treatment with bis(7)-tacrine prevented the decline of ∆ψm (Figure 1A) and the decrease of ATP levels and cell death induced by glutamate in a concentration-dependent manner (Figure 2B). These results indicate that post-treatment with bis(7)-tacrine may exert its neuroprotection via maintaining mitochondrial function and/or its downstream signaling pathways instead of the blockade of NMDA receptors. Because bis(7)-tacine is a potent AChE inhibitor, we considered if AChE inhibition is involved in this neuroprotection. However, we found that the neuroprotection of bis(7)-tacrine was independent of AChE inhibition from the following findings. Pretreatment with tacrine and E2020 (donepezil), two well-known AChE inhibitors for AD, failed to prevent glutamate-induced excitotoxicity even at concentrations that have a similar inhibition of AChE activity as 1 µM bis(7)-tacrine.15 Furthermore, post-treatment with tacrine or E2020 did not prevent the decline of ∆ψm (Figure 1B), the decrease of ATP levels, and cell death induced by glutamate in CGNs (Figure 2C). In addition, the activation of cholinergic receptors has been shown to prevent the excitotoxicity induced by glutamate.22-24 Some selective AChE inhibitors, such as rivastigmine and galantamine, are able to directly activate cholinergic receptors and affect the glutamatergic system in mammalian central nervous systems.25,26 However, atropine (a muscarinic receptor antagonist), dihydro-β-erythroidine (a nicotinic cholinergic receptor antagonist), or a combination of both failed to attenuate the neuroprotection of bis(7)-tacrine against glutamate-induced excitotoxicity in CGNs (data not shown). These results suggest that the neuroprotection against glutamate-induced mitochondrial dysfunction and cell death by post-treatment with bis(7)-tacrine is independent of NMDA antagonism, AChE inhibition, and cholinergic pathways in our system. Mitochondrial Isolation and Purity Identification. It is wellknown that high-purity mitochondrial preparation is a critical step for the reliabilities of proteomic experiments.27 This study followed a protocol for the isolation and characterization of intact, functional mitochondria from primary rat CGN cultures as described in Materials and Methods. All the mitochondrial separations were performed with a common isolation method, including (1) gentle cell disruption via homogenization, (2) crude mitochondrial separation by differential centrifugation, and (3) recovery of highly purified organelles using Percoll centrifugation.18 The purity of mitochondrial, cytosolic, and nuclear fractions was verified using Western blot analysis with specific COXIV (a mitochondria-specific protein), Histone H2A (a nuclear marker), and Raf-1 (a cytosolic marker) antibodies. Our data showed that there was little cross-contamination between different fractions with the use of our subcellular fractionation method (Figure 3). Comparative Analysis of Mitochondrial Proteomes by 2-D DIGE and Protein Identification. The shortcoming of traditional two-dimensional gel electrophoresis is not being able to reflect gel-to-gel variations in the spot patterns. 2-D DIGE,

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Figure 3. The purity identification of subcellular fractions from CGNs. Western blot analyses of mitochondrial (mito, lane 1), cytosolic (cyto, lane 2), and nuclear fractions (nucl, lane 3) were probed for COXIV (a mitochondria-specific protein), Histone H2A (a nuclear marker), and Raf-1 (a cytosolic marker). These data showed that there was little cross-contamination between different fractions using our subcellular fractionation method. This is a representative result from three independent experiments.

a multiplexing-based approach with an internal pool standard, allows to circumvent the variability that often shades biological differences or misleads quantitative comparisons of protein expression levels.28 To investigate the intracellular mitochondria-associated mechanisms of bis(7)-tacrine against glutamate, 2-D DIGE was used to analyze the differentiation of mitochondrial proteomes between CGNs cultures treated with the vehicle alone, with glutamate, and with glutamate plus post-treatment with bis(7)-tacrine. Mitochondria from these treatment groups were isolated and differentially labeled with Cy3 and Cy5. An internal standard composed of each mitochondrial sample and labeled with Cy2 was added to improve the comparative analysis (Table 1 in Supporting Information). The labeled samples were mixed and co-separated on broad range pH 310 NL 2-D gels. The gels were then scanned in a wavelengthselective way, and subsequent overlaid 2-D DIGE images codetected by Typhoon 9400 (Figure 1 in Supporting Information) were analyzed with the Decyder differential analysis software. Figure 4 showed the characteristic mitochondrial protein pattern containing equal amounts of mitochondrial proteins from each group. In theory, the standardized logarithm values of protein abundances follow a normal distribution and are comparable across all spots and gels. Therefore, at present, the standard Student’s t test in the DeCyder software is one of the most popularly used methods for statistically analyzing the differentiation of protein abundances between two treated groups based on the standardized logarithm (i.e., log) values of protein abundances.29 According to statistical tests of the 2-D DIGE gels (Student’s t test, p < 0.01), 84 of the overall 1190 protein spots exhibited differences in the normalized spot volume ratios exceeding 20%. Among them, 47 proteins were identified by searching the NCBInr protein database (Tables 1 and 2), including 29 nonredundant proteins, which are mainly involved in three biological processes including energy metabolism, oxidative stress, and apoptosis. Furthermore, the changes in 22 of these nonredundant proteins were reversed by posttreatment with bis(7)-tacrine. However, Student’s t test is not always valid for large-scale protein expression data. Some researchers have pointed out the statistical challenges in the analysis of proteomic data using the DeCyder software.29,30 Solid statistics will yield higher confidence in experimental findings and promote the development of proteomics.31 Novel statistical methods for large-scale proteomic data are needed. Effects of Bis(7)-tacrine on the Changes in Energy Metabolism-Associated Proteins after Glutamate Treatment. MitoJournal of Proteome Research • Vol. 6, No. 7, 2007 2439

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Figure 4. Representative Deep Purple post-stained preparative gel loaded with a mixture of mitochondrial extracts from three different treatment groups. At 8 DIV, CGNs were exposed to 75 µM glutamate, and then 1 µM bis(7)-tacrine was added 1 h later. After 3 h of glutamate challenge, mitochondria from different treatment groups were isolated, and 166.67 µg of each mitochondrial sample was pooled together. The pooled samples were loaded on 24 cm pH 3-10 NL IPG-strips and subjected to isoelectrofocusing and seconddimension electrophoresis in 12.5% SDS-PAGE gels. After 2-D electrophoresis, the preparative gels were post-stained with Deep Purple and scanned using a Typhoon 9400 scanner at the excitation wavelength of 532 nm. The image generated was then matched to the analytical gel set. The proteins that were found to vary above 1.2-fold (p < 0.01, Student’s t test) in analytical gels labeled with CyDyes were marked with the number allocated by the DeCyder software in the preparative gel post-stained with Deep Purple.

chondria, the energy plants of cells, generate ATP through mitochondrial electron transport chain (ETC) coupled with oxidative phosphorylation (OXPHOS). This respiratory-chain consists of electron acceptors, coenzyme Q, and cytochrome c, and five multisubunit protein complexes (complex I-V).32 Deficiency in complex I function has been associated with neurodegenerative diseases and the aging process.32,33 Furthermore, glutamate has been shown to inhibit respiration and sequentially decrease the ATP production.2,34 In this study, it also showed that some subunits or precursors of complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome bc1), complex IV (cytochrome c oxidase), and complex V (ATP synthetase) dramatically decreased in mitochondria from CGNs exposed to glutamate for 3 h; and the changes in complex I, IV, and V could be significantly reversed by post-treatment with bis(7)-tacrine (Table 1). In addition, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which plays a pivotal role in carbohydrate metabolism, increased by 30-40% in mitochondria treated with glutamate when compared with the control group (Table 1). There are two possible reasons for this increase: (1) it is a compensatory response to increase ATP production via glycolysis by some surviving or apoptotic neurons in the presence of glutamate-induced respiration inhibition; or (2) since people have recently found that translocation of GAPDH to nuclei and/ or mitochondria may play an important role in cytosine arabinoside- and low potassium-induced apoptosis in CGNs,35,36 GAPDH translocates to mitochondria during glutamate2440


induced cell death is another possibility. The GAPDH protein levels declined significantly when CGNs were post-treated with bis(7)-tacrine (Table 1). Mitochondrial aconitase 2, an isomerase that converts citrate into isocitrate in the TCA cycle, also increased significantly during glutamate treatment, while bis(7)-tacrine dramatically reversed this change (Table 1). However, decreased mitochondrial aconitase activity could be detected during the exposure of NMDA in cortical neurons and used as an effective marker for mitochondrial reactive oxygen species (ROS) detection in excitotoxic cell death.37,38 The increase in glutamate-induced mitochondrial aconitase 2 may be a compensatory response to enhance ATP production via the TCA cycle or a defense mechanism against ROS-induced oxidative stress resulting from glutamate-caused respiration inhibition. Furthermore, a recent study has identified a second function for aconitase in stabilizing mitochondrial DNA (mtDNA) from the oxidative stress environment.39 Why bis(7)-tacrine reversed the increase in mitochondrial aconitase 2 requires further study. Reversal of Glutamate-Induced Changes in Oxidative StressAssociated Proteins by Bis(7)-tacrine. ROS- and reactive nitrogen species (RNS)-induced mitochondrial dysfunction plays an important role in glutamate excitotoxicity.7,40 ROS attack polyunsaturated fatty acids and lead to membrane lipid peroxidation, thereby generating reactive aldehydes including 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde, which is a key mediator of neuronal cell death induced by oxidative insults and is mainly detoxified by mitochondrial aldehyde

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Table 1. List of Proteins Exhibiting Different Abundances in Mitochondria Proteins from CGNs Exposed to Glutamate and Post-Treated with or without Bis(7)-tacrinea category

spot no.

protein name

t test

G/CT

t test

GBT/G

-1.56 -1.66 -1.31 -1.6 -1.51 -1.61 -1.48 -1.43 -1.48 -1.44 -1.44 -1.48 -1.42 -1.5 -1.43 -1.44 -1.38 -1.38 -1.4 -1.35 -1.4 -1.49 -1.43

2.50E-06 3.30E-05 4.30E-07 0.0018 7.40E-06 1.10E-06 0.0029 7.10E-07 0.0052 6.00E-06 0.0016 0.00075 1.30E-08 4.30E-05 1.40E-08 0.021 0.41 0.00096 7.00E-05 0.002 0.0022 1.90E-08 0.0001

2.34 1.75 -1.93 -1.25 -1.40 -1.59 1.38 1.38 1.38 1.34 1.36 1.31 1.47 1.35 1.61 1.06 1.06 -1.21 1.47 1.27 2.09 1.58 1.65

1.3 1.44

0.0049 0.0070

-1.34 -1.20

1.21

0.00088

-2.00

Respiratory chain

222 229 318 322 323 324 466 467 470 474 484 487 539 547 557 723 929 972 995 1019 1039 1042 1146

NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75 kDa NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75 kDa succinate dehydrogenase complex, subunit A, flavoprotein succinate dehydrogenase complex, subunit A, flavoprotein succinate dehydrogenase complex, subunit A, flavoprotein succinate dehydrogenase complex, subunit A, flavoprotein ATP synthase alpha subunit precursor ATP synthase alpha subunit precursor ATP synthase alpha subunit precursor ATP synthase alpha subunit precursor ATP synthase alpha subunit precursor ATP synthase alpha subunit precursor ATP synthase beta subunit ATP synthase beta subunit ATP synthase beta subunit NADH dehydrogenase 1 alpha subcomplex 10-like protein succinate dehydrogenase complex subunit B Rieske Fe-S protein precursor 24-kDa mitochondrial NADH dehydrogenase precursor similar to NADH dehydrogenase Fe-S protein 8 ATP synthase, mitochondrial F0 complex, subunit d ATP synthase, mitochondrial F0 complex, subunit d cytochrome c oxidase subunit VIa (AA 1-118)

0.0049 7.90E-05 3.30E-05 4.30E-11 2.10E-06 0.00032 0.0013 4.10E-07 0.0092 9.90E-06 0.0045 0.00024 2.30E-07 8.00E-06 1.40E-05 3.10E-05 0.0058 9.40E-05 0.00036 2.20E-05 0.002 9.90E-09 0.00066

Carbohydrate metabolism

752 775

glyceraldehyde-3-phosphate dehydrogenase glyceraldehyde-3-phosphate dehydrogenase

0.0025 0.0023

TCA cycle

177

aconitase 2, mitochondrial

0.0015

Oxidative stress and detoxification

543 992

mitochondrial aldehyde dehydrogenase glyoxylase 1

0.0028 0.00017

-1.36 -1.38

0.0013 1.00E-05

1.32 1.92

Channel protein

841 873

voltage-dependent anion channel 1 (Vdac1) voltage-dependent anion channel 2 (Vdac2)

3.50E-07 0.0012

-1.48 -1.48

0.0023 0.6

1.39 1.03

Chaperone and/or Signaling

910 925 174 175 162 814

similar to prohibitin 14-3-3 β heat shock protein 1, alpha heat shock protein 1, alpha optic atrophy 1-like protein guanine nucleotide-binding protein, beta-1 subunit

0.00057 7.40E-05 0.00031 0.0046 0.005 0.0081

-1.42 1.3 1.68 1.44 -1.65 1.26

9.80E-07 2.10E-06 0.00021 0.0076 0.00011 1.30E-06

1.67 -1.99 -2.04 -1.75 -1.42 -2.22

Cytoskeleton protein

609 663 664 665 445 454 561

actin beta actin beta actin beta actin beta tubulin alpha tubulin, alpha 2 tubulin beta chain 15

0.00054 0.0065 1.00E-05 5.70E-06 0.0014 0.00014 0.0079

1.31 1.22 1.47 1.59 1.42 1.6 1.24

6.40E-06 9.80E-05 8.70E-06 8.30E-08 5.90E-06 0.00086 8.70E-06

-2.08 -1.66 -3.06 -3.14 -3.73 -2.22 -2.11

Mitochondiral related proteins

850 893 125 790

holocytochrome c-type synthase complement component 1, q subcomponent binding protein hypothetical protein LOC314432 similar to capping protein muscle z-line, alpha 1

0.0024 0.0034 0.00015 0.0077

0.17 0.0011 0.00065 0.00013

-1.13 -2.02 -2.21 1.98

-1.59 1.22 1.37 -1.33

a Proteins that significantly varied in the glutamate treated group (G) versus control (CT) (G/CT > 1.2, p < 0.01, Student’s t test) are categorized according to their general function. Legend abbreviations: Spot no., spot number allocated by the DeCyder software; t test, value of the Student’s t test; GBT, CGNs were exposed to glutamate and bis(7)-tacrine was added 1 h later. G/CT and GBT/G, amplitude of variation where a positive value means that the amount of protein is increased.

dehydrogenase 2 (ALDH2). ALDH2 deficiency has been shown to enhance oxidative stress and increase the risk for late-onset AD.41,42 Decreased in situ activity of glyoxalase I, another oxdative stress and detoxification associated protein, due to the aging process and oxidative stress results in an increase in glycation and tissue damage.43 In the present study, the protein levels of mitochondrial aldehyde dehydrogenase and glyoxylase I, that is, glyoxalase I, decreased by 36% and 38%, respectively, in glutamate-treated group, while bis(7)-tacrine reversed these decreases (Table 1). These results suggest that glutamateinduced oxidative stress may impact the expression and activity of mitochondrial aldehyde dehydrogenase and glyoxalase I. Effects of Bis(7)-tacrine on Changes in Apoptosis-Associated Proteins after Glutamate Treatment. Increasing evidence

suggests that the movement of key proteins in or out of mitochondria during apoptosis is essential for the regulation of apoptosis and translocation of BAD, the transcription factor TR3, the Peutz-Jegher gene product LKB1, and the actinbinding protein cofilin to mitochondria all of which have been shown to induce apoptosis.44 Voltage-dependent anion channel (VDAC), together with adenine nucleotide translocator (ANT) and cyclophilin D, controls the opening of the mitochondrial PTP and subsequent release of cytochrome c, apoptosisinducing factor ,and some procaspases that lead to apoptotic cell death.45 However, it has been argued that the closure of VDAC, not the opening, leads to the outer membrane permeabilization of mitochondria and apoptosis.46 Furthermore, VDAC2, an isoform of VDAC, can inhibit mitochondrial apoJournal of Proteome Research • Vol. 6, No. 7, 2007 2441

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Table 2. Results of MALDI-TOF/MS and NCBInr Protein Database Searching for Protein Identificationa spot no.

protein name

AC (gi)

MW

pI

NP

C

MS

222 229 318 322 323 324 466 467 470 474 484 487 539 547 557 723 929 972 995 1019 1039 1042 1146

NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75 kDa NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75 kDa succinate dehydrogenase complex, subunit A, flavoprotein succinate dehydrogenase complex, subunit A, flavoprotein succinate dehydrogenase complex, subunit A, flavoprotein succinate dehydrogenase complex, subunit A, flavoprotein ATP synthase alpha subunit precursor ATP synthase alpha subunit precursor ATP synthase alpha subunit precursor ATP synthase alpha subunit precursor ATP synthase alpha subunit precursor ATP synthase alpha subunit precursor ATP synthase beta subunit ATP synthase beta subunit ATP synthase beta subunit NADH dehydrogenase 1 alpha subcomplex 10-like protein succinate dehydrogenase complex subunit B Rieske Fe-S protein precursor 24-kDa mitochondrial NADH dehydrogenase precursor similar to NADH dehydrogenase Fe-S protein 8 ATP synthase, mitochondrial F0 complex, subunit d ATP synthase, mitochondrial F0 complex, subunit d cytochrome c oxidase subunit VIa (AA 1-118)

53850628 53850628 18426858 18426858 18426858 18426858 203055 203055 203055 203055 203055 203055 1374715 1374715 1374715 32996721 89573817 206681 205628 27661165 9506411 9506411 55992

80331 80331 72596 72596 72596 72596 58904 58904 58904 58904 58904 58904 51171 51171 51171 40804 27377 27956 26854 24411 18809 18809 12966

5.65 5.65 6.75 6.75 6.75 6.75 9.22 9.22 9.22 9.22 9.22 9.22 4.92 4.92 4.92 7.14 8.33 8.9 6 5.87 6.17 6.17 6.46

8 16 14 13 10 20 9 7 19 12 10 9 7 13 13 6 9 5 10 9 7 8 8

15% 28% 31% 30% 20% 39% 22% 17% 40% 31% 21% 17% 19% 39 39 26% 41% 11% 38% 41% 58% 58% 41%

109 207 193 164 142 227 118 111 217 146 119 126 83 202 109 108 133 65 120 115 99 124 81

752 775

glyceraldehyde-3-phosphate dehydrogenase glyceraldehyde-3-phosphate dehydrogenase

8393418 8393418

36090 36090

8.14 8.14

5 5

12% 12%

64 64

177

aconitase 2, mitochondrial

40538860

86121

7.87

8

13%

94

543 992

mitochondrial aldehyde dehydrogenase glyoxylase 1

25990263 46485429

53809 20977

5.7 5.12

9 5

25% 23%

116 65

841 873

voltage-dependent anion channel 1 (Vdac1) voltage-dependent anion channel 2 (Vdac2)

38051979 13786202

32060 32353

8.35 7.44

11 8

51% 36%

168 129

910 925 174 175 162 814

similar to prohibitin 14-3-3 β heat shock protein 1, alpha heat shock protein 1, alpha optic atrophy 1-like protein guanine nucleotide-binding protein, beta-1 subunit

62664759 9507243 28467005 28467005 37812499 13591874

27757 28151 85161 85161 111737 38161

5.44 4.81 4.93 4.93 7.17 5.47

8 8 16 11 9 6

43% 27% 23% 17% 13% 15%

132 71 139 115 90 67

609 663 664 665 445 454 561

actin beta actin beta actin beta actin beta tubulin alpha tubulin, alpha 2 tubulin beta chain 15

71620 71620 71620 71620 223556 34740335 92930

42066 42066 42066 42066 50894 50804 50361

5.29 5.29 5.29 5.29 4.94 4.94 4.79

9 7 10 6 14 11 7

32% 23 32% 25% 38% 35% 15%

105 95 120 105 146 116 91

850 893 125 790

holocytochrome c-type synthase complement component 1, q subcomponent binding protein hypothetical protein LOC314432 similar to capping protein muscle z-line, alpha 1

109510612 48675371 62078893 109465391

31452 31320 118910 33060

6.25 4.77 5.36 5.43

6 5 6 9

26% 15% 8% 46%

100 59 61 136

a Results of MALDI-TOF mass spectra and protein identification through searching NCBI nonredundant database. Legend abbreviations: AC (gi), access number of gene bank ID; MW, molecular weight; pI, isoelectric point; NP, number of matched peptides; C, sequence coverage; MS, Mowse score.

ptosis by binding and regulating the activity of the proapoptotic protein Bak.47 In this study, both VDAC1 (Table 1 and Figure 5B) and VDAC2 (Table 1) were down-regulated by 48% after a 3 h challenge with glutamate in CGNs, while only the VDAC1 expression level was reversed by bis(7)-tacrine. Glutamate-induced down-regulation of VDAC1 may be due to inhibition of VDAC activity by high NADH levels during glutamate treatment or due to the closure of VDAC, not the opening, leading to glutamate-induced apoptosis. Downregulation of VDAC2 by glutamate verifies the anti-apoptotic role of VDAC2. The prohibitins (prohibitin1 and prohibitin2) form a high molecular weight complex in the mitochondrial inner membrane and stabilize the newly synthesized subunits of mitochondrial respiratory enzyme.48 The expression of the prohibitins changes with senescence, which suggests that a decline 2442


in prohibitins might be associated with the accumulation of damage from mitochondrial oxygen radicals.49 In our study, the prohibitin level in the glutamate-treated mitochondria decreased by 42% when compared with the control group, while bis(7)-tacrine could significantly up-regulate the prohibitin level (Table 1 and Figure 5A). 14-3-3 proteins are abundantly expressed in the brain and have been detected in the cerebrospinal fluid of patients with different neurological disorders. Recent evidence indicates that 14-3-3, by interacting with binding partners, may be involved in the inhibition of ATP synthase activity,50 apoptosis,51,52 and mitochondrial protein assembly.53 As shown in Table 1 and Figure 5C, compared with the control group, the protein level of 14-3-3 β increased by 30% in mitochondria treated with glutamate, whereas bis(7)tacrine dramatically decreased the level of 14-3-3 β, even below the level of the control group.


research articles

Figure 5. Comparative analysis of four protein spot intensities using the BVA module of the DeCyder software. The selected four protein spots of different treatment groups are displayed as three-dimensional images (top panels) and as partial views of the 2D-gel (bottom panels). The spot boundary of selected proteins is displayed in pink.

The heat shock protein 1R (Hsp90) is a molecular chaperone that assists both in ATP-independent sequestration of damaged proteins and in ATP-dependent folding of numerous targets, such as nuclear hormone receptors and protein kinases.54 Hsp90 can form a novel complex with soluble guanylate cyclase (sGC) and NOS, and it has been hypothesized that these complexes serve to target intracellular NO produced by NOS directly to sGC and, thus, prevent inactivation of NO by reaction with superoxide anion and production of the highly toxic molecule peroxynitrite.55 In this study, the significant increase in the Hsp90 protein level in mitochondria exposed to glutamate (Table 1 and Figure 5D) suggests that this might be a compensatory response to glutamate-induced oxidative stress. However, the reason bis(7)-tacrine reversed this increase in the Hsp90 protein level remains to be examined. Optic atrophy 1-like protein (OPA1) is a dynamin-like GTPase of the mitochondrial intermembrane space important for maintaining cristae structure. OPA1 slows down cytochrome c release by maintaining tight cristae, and the levels of OPA1 decline significantly during apoptosis.56,57 In our model, OPA1 was significantly down-regulated during glutamate-induced apoptosis (Table 1), which could not be reversed by bis(7)tacrine. In addition, the level of guanine nucleotide-binding protein beta, which functions as a modulator in various transmembrane signaling pathways and is required for GTPase activity, showed a significant 26% increase in mitochondria from glutamate-treated neurons (Table 1), and it was dramatically down-regulated by post-treatment with bis(7)-tacrine. Glutamate has also been shown to stimulate GTP binding.58 The real relationship between the elevation of guanine nucleotide binding protein beta and glutamate-induced cell death is worthy of further study.

It is noteworthy that the expression of two cytoskeleton proteins, tubulin and actin, increased significantly after glutamate challenge, and this was reversed by bis(7)-tacrine (Table 1). Recent evidence suggests that the actin cytoskeleton has a role in regulating apoptosis via the interaction with mitochondria, which also appears to have a significant impact on the management of oxidative stress and cellular aging.59 Furthermore, mitochondrial accumulation of β-actin occurs before the mitochondrial insertion of Bax and the release of cytochrome c in apoptosis, and this suggests that actin can contribute to the initiation of apoptosis by enabling cytosolic pro-apoptotic proteins to be carried to mitochondria by the cytoskeletondriven trafficking system.60 Tubulin, another important cytoskeleton protein, has been recently identified in the nucleus of cells and in mitochondria. Cellular tubulin is reorganized into visible tubulin structures that correlate with the apoptotic morphology, the level of tubulin mRNA displays a transient increase during apoptosis, and downstream events from tubulin binding are believed to be critical events for the generation of apoptosis.61,62 However, the exact reasons for elevations of actin and tubulin in glutamate-treated neurons require further investigation. Effects of Bis(7)-tacrine on Changes in Other Mitochondrial Proteins Induced by Glutamate. Mitochondrial holocytochrome c-type synthase (Hccs) catalyzes the covalent attachment of heme to both apocytochrome c and c1, the precursor forms, thereby leading to the mature forms, holocytochrome c and c1, which are necessary for proper functioning of the mitochondrial respiratory chain. In addition to the well-known role of cytochrome c in OXPHOS, cytochrome c is released from mitochondria in response to a variety of intrinsic deathpromoting stimuli that, in turn, result in apoptosis.63 Therefore, Journal of Proteome Research • Vol. 6, No. 7, 2007 2443

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Figure 6. Western blot analysis of the expression of prohibitin, VDAC1, 14-3-3 β, Hsp90, and COXIV. Mitochondrial and cytosolic proteins from each group were separated by 12.5% SDS-PAGE. The proteins were transferred to PVDF membranes, then probed with antibodies against prohibitin, VDAC1, 14-3-3 β, Hsp90, and COXIV, and visualized with an ECL Western blot detection kit. This is a representative result from three independent experiments.

the significant decrease of Hccs in glutamate-treated neurons (Table 1) may be due to two reasons. The first reason is that glutamate tries to inhibit the respiration of neurons via direct inhibition of the expression and/or activity of Hccs; and the other is that this decrease is a compensatory response to inhibit glutamate-induced apoptosis via reducing the production of mature cytochrome c. However, bis(7)-tacrine does not reverse this decrease (Table 1). Complement component 1, q subcomponent binding protein (Clq) plays a key role in the recognition of immune complexes, thereby initiating the classical complement pathway. It is possible that the Clq-Clq receptor interaction is crucial in down-regulation of apoptosis via clearance of apoptotic bodies.64 In this study, we found that bis(7)-tacrine significantly reduced the expression of C1q enhanced by glutamate. Thus, the elevation of Clq might be a compensatory response to remove the apoptotic bodies produced by glutamate. In this study, two proteins with unknown function were also identified from our 2D-gel. Hypothetical protein LOC314432 increased in mitochondria after glutamate treatment, whereas the expression of the other protein, similar to capping protein muscle z-line, alpha 1 protein, showed a significant decrease. These changes were reversed by bis(7)-tacrine, which suggests that they might also be possible biomarkers for glutamate neurotoxicity and for the neuroprotection of bis(7)-tacrine. These observations also highlight the great advantages of the proteomic technique in probing the whole cellular or subcellular proteomes simultaneously and in finding new functional proteins. Western Blot Analysis of Four Interesting Proteins. To test the veracity of the proteomic results, four proteins in which we are interested, including prohibitin, VDAC1, 14-3-3 β, and Hsp90, were selected and analyzed by Western blot (Figure 6). 2444


The expression levels of COXIV and Raf-1, which showed few changes, were used as loading controls for mitochondrial and cytosolic fractions, respectively. Compared with the control group, in the glutamate-treated CGNs, the levels of prohibitin and VDAC1 protein in the mitochondrial fractions decreased significantly, whereas the levels of 14-3-3 β and Hsp90 increased dramatically. As expected, bis(7)-tacrine could reverse the glutamate-induced changes in these four proteins in mitochondrial fractions accordingly. The expression levels of these four proteins in the cytosolic fractions showed no significant differences among these three groups. The changes of these four proteins in the mitochondrial fractions were consistent with our 2-D DIGE results (Figure 5), confirming the veracity of the 2-D DIGE gel image data and providing a rationale for future functional studies of these proteins.

Conclusion In this study, we found that bis(7)-tacrine reversed declines in mitochondrial membrane potential, ATP production, and neuronal cell death caused by treatment with glutamate. More importantly, 2-D DIGE analysis showed that bis(7)-tacrine reversed the expression patterns in 22 out of 29 proteins after glutamate insult. These changes are mainly in proteins involved in energy metabolism, oxidative stress, and apoptosis. The changes of four interesting proteins were further validated using Western blot. Our findings suggest that multiple signaling pathways initiated by altered mitochondrial proteins might mediate glutamate-induced excitotoxicity and also provide potentially useful intracellular targets for the neuroprotection of bis(7)-tacrine. Combined, these results with previous studies, we postulate that post-treatment of bis(7)-tacrine might prevent glutamate excitotoxicity by maintaining normal mitochondrial function, targeting mitochondrion-associated apoptotic signal-


ing pathways, and inhibiting the activity of NOS and oxidative stress induced by glutamate (see TOC synopsis), which may offer a novel direction in rational development of beneficial agents for the treatment of neurodegenerative diseases. Abbreviations: 2-D DIGE, two-dimensional differential ingel electrophoresis; CGNs, cerebellar granule neurons; PTP, permeability transition pore; OXPHOS, oxidative phosphorylation; ROS, reactive oxygen species; VDAC, voltage-dependent anion channel; Hsp90, heat shock protein 1, alpha; NOS, nitric oxide synthase; OPA1, optic atrophy 1-like protein; Hccs, holocytochrome c-type synthase.

Acknowledgment. This work was supported by grants from the Research Grants Council of Hong Kong (HKUST6441/ 06M, HKUST6133/03M, DAG06/07M, and AoE/B15/01) and the National Science Foundation of China (30370450, 30170299, and 30570562). We thank Dr. Virginia A. Unkefer for proofreading our manuscript. Supporting Information Available: Experimental design for the 2-D DIGE method and analysis of mitochondrial protein patterns from three different treatment groups. This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) Choi, D. W. Calcium and excitotoxic neuronal injury. Ann. N. Y. Acad. Sci. 1994, 747, 162-171. (2) Rego, A. C.; Santos, M. S.; Oliveira, C. R. Glutamate-mediated inhibition of oxidative phosphorylation in cultured retinal cells. Neurochem. Int. 2000, 36, 159-166. (3) Kushnareva, Y. E.; Wiley, S. E.; Ward, M. W.; Andreyev, A. Y.; Murphy, A. N. Excitotoxic injury to mitochondria isolated from cultured neurons. J. Biol. Chem. 2005, 280, 28894-28902. (4) Choi, D. W. Glutamate neurotoxicity and diseases of the nervous system. Neuron 1988, 1, 623-634. (5) Tymianski, M.; Charlton, M. P.; Carlen, P. L.; Tator, C. H. Source specificity of early calcium neurotoxicity in cultured embryonic spinal neurons. J. Neurosci. 1993, 13, 2085-2104. (6) Schinder, A. F. Mitochondrial dysfunction is a primary event in glutamate neurotoxicity. J. Neurosci. 1996, 16, 6125-6133. (7) Nicholls, D. G. Mitochondrial dysfunction and glutamate excitotoxicity studied in primary neuronal cultures. Curr. Mol. Med. 2004, 4, 149-177. (8) Khodorov, B. I. Mechanisms of destabilization of Ca2+-homeostasis of brain neurons caused by toxic glutamate challenge. Membr. Cell Biol. 2000, 14, 149-162. (9) Krieger, C.; Duchen, M. Mitochondria, Ca2+ and neurodegenerative disease. Eur. J. Pharmacol. 2002, 447, 177-188. (10) Wang, H.; Carlier, P. R.; Ho, W. L.; Wu, D. C.; Lee, N. T.; Li, C. P.; Pang, Y. P.; Han, Y. F. Effects of bis(7)-tacrine, a novel antiAlzheimer’s agent, on rat brain AChE. Neuroreport 1999, 10, 789793. (11) Liu, J.; Ho, W.; Lee, N. T.; Carlier, P. R.; Pang, Y.; Han, Y. Bis(7)tacrine, a novel acetylcholinesterase inhibitor, reverses AF64Ainduced deficits in navigational memory in rats. Neurosci. Lett. 2000, 282, 165-168. (12) Wu, D. C.; Xiao, X. Q.; Ng, A. K.; Chen, P. M.; Chung, W.; Lee, N. T.; Carlier, P. R.; Pang, Y. P.; Yu, A. C.; Han, Y. F. Protection against ischemic injury in primary cultured mouse astrocytes by bis(7)tacrine, a novel acetylcholinesterase inhibitor. Neurosci. Lett. 2000, 288, 95-98. (13) Xiao, X. Q.; Lee, N. T.; Carlier, P. R.; Pang, Y. P.; Han, Y. F. Bis(7)-tacrine, a promising anti-Alzheimer’s agent, reduces hydrogen peroxide-induced injury in rat pheochromocytoma cells: comparison with tacrine. Neurosci. Lett. 2000, 290, 197-200. (14) Fu, H.; Li, W.; Lao, Y.; Luo, J.; Lee, N. T.; Kan, K. K.; Tsang, H. W.; Tsim, K. W.; Pang, Y.; Li, Z.; Chang, D. C.; Li, M.; Han, Y. Bis(7)tacrine attenuates beta amyloid-induced neuronal apoptosis by regulating L-type calcium channels. J. Neurochem. 2006, 98, 1400-1410.

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