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Brain Research 1044 (2005) 206 – 215 www.elsevier.com/locate/brainres

Research report

Proteomic identification of proteins oxidized by Ah(1–42) in synaptosomes: Implications for Alzheimer’s disease Debra Boyd-Kimballa, Alessandra Castegnaa, Rukhsana Sultanaa, H. Fai Poona, Robin Petrozea, Bert C. Lynna,b, Jon B. Kleind, D. Allan Butterfielda,c,* a

Department of Chemistry, Center for Membrane Sciences, University of Kentucky, Lexington, KY 40506-0055, USA b Core Proteomics Laboratory, University of Kentucky, Lexington, KY 40506-0055, USA c Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40506-0055, USA d Kidney Disease Program and Proteomics Core Laboratory, University of Louisville School of Medicine and VAMC, Louisville, KY 40292, USA Accepted 24 February 2005 Available online 15 April 2005

Abstract Protein oxidation has been implicated in Alzheimer’s disease (AD) and can lead to loss of protein function, abnormal protein turnover, interference with cell cycle, imbalance of cellular redox potential, and eventually cell death. Recent proteomics work in our laboratory has identified specifically oxidized proteins in AD brain such as: creatine kinase BB, glutamine synthase, ubiquitin carboxy-terminal hydrolase L-1, dihydropyrimidase-related protein 2, a-enolase, and heat shock cognate 71, indicating that a number of cellular mechanisms are affected including energy metabolism, excitotoxicity and/or synaptic plasticity, protein turnover, and neuronal communication. Synapse loss is known to be an early pathological event in AD, and incubation of synaptosomes with amyloid beta peptide 1 – 42 (Ah 1 – 42) leads to the formation of protein carbonyls. In order to test the involvement of Ah(1 – 42) in the oxidation of proteins in AD brain, we utilized two-dimensional gel electrophoresis, immunochemical detection of protein carbonyls, and mass spectrometry to identify proteins from synaptosomes isolated from Mongolian gerbils. Ah(1 – 42) treatment leads to oxidatively modified proteins, consistent with the notion that Ah(1 – 42)-induced oxidative stress plays an important role in neurodegeneration in AD brain. In this study, we identified h-actin, glial fibrillary acidic protein, and dihydropyrimidinase-related protein-2 as significantly oxidized in synaptosomes treated with Ah(1 – 42). Additionally, H+-transporting two-sector ATPase, syntaxin binding protein 1, glutamate dehydrogenase, g-actin, and elongation factor Tu were identified as increasingly carbonylated. These results are discussed with respect to their potential involvement in the pathogenesis of AD. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Degenerative disease: Alzheimer’s-beta amyloid Keywords: Alzheimer’s disease; Amyloid h-peptide (1 – 42); Amyloid h-peptide (42 – 1); Oxidative stress; Proteomics

1. Introduction Alzheimer’s disease (AD) is characterized pathologically by the presence of senile plaques, neurofibrillary tangles, * Corresponding author. Department of Chemistry, Center for Membrane Sciences, and Sanders-Brown Center on Aging, 121 ChemistryPhysics Building, University of Kentucky, Lexington, KY 40506-0055, USA. Fax: +1 859 257 5876. E-mail address: [email protected] (D.A. Butterfield). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.02.086

and synapse loss. Additionally, AD is associated with oxidative stress. Amyloid beta peptide is a 39 –43 amino acid peptide produced from proteolytic processing of amyloid precursor protein (APP), a ubiquitous transmembrane glycoprotein [58]. Ah(1 –42) is the primary component of the core of senile plaques and has been shown to induce protein oxidation in vitro and in vivo [6 – 8,14,28,34,41,49,69]. Ah(1– 42)-induced oxidative stress has been proposed to play a central role in the pathogenesis of AD [8,10,14 – 16]. Consistent with this notion, oxidative

D. Boyd-Kimball et al. / Brain Research 1044 (2005) 206 – 215

stress in AD brain has been shown to be region-specific in correlation with the presence of Ah(1 –42) [34]. Protein oxidation is increased in brain regions that are rich in Ah(1– 42) such as the hippocampus and cortex but not in regions poor in Ah(1– 42) such as the cerebellum [34]. Recent studies from our laboratory have utilized proteomic techniques to identify proteins that are specifically oxidized in AD brain versus age-matched control [10,17,19 –21]. These techniques involve the use of a parallel analysis between a two-dimensional polyacrylamide gel (2DPAGE) and oxyblot (i.e., 2D-PAGE transferred to a nitrocellulose membrane and developed immunochemically for protein carbonyls, a marker of protein oxidation [9,14]) to identify protein spots which are increased in carbonyl reactivity. In the inferior parietal lobule (IPL) creatine kinase BB, glutamine synthetase, ubiquitin carboxy-terminal hydrolase L-1, dihydropyrimidinase-related protein-2, a-enolase, and heat shock cognate 71 were found to be significantly oxidatively modified in AD brain compared to control. There is evidence that protein oxidation leads to conformational changes in protein structure that result in loss of protein function [34,40,59]. Based on this notion, the proteins found to be oxidized in AD brain affect a wide variety of cellular functions including energy metabolism, glutamate uptake and excitotoxicity, proteosome function, neuritic connections, and neuronal communications consistent with the pathological findings associated with the disease [10 –13,17]. Synaptic alterations have been shown to be an early event in the pathogenesis of AD [26,32,36,47]. In particular, it is believed that early synaptic loss in the hippocampal dentate gyrus disrupts the circuitry between the hippocampus and the entorhinal cortex leading to the memory deficits associated with AD [44]. Moreover, damage in these regions corresponds with severity of dementia, oxidative stress, and deposition of Ah(1 – 42) [27,34,60]. Ah(1– 42) added to synaptosomes leads to protein oxidation [40,41,47]. Based on these findings, we reasoned that proteomic investigation of protein oxidation induced by Ah(1– 42) on synaptosomes could provide insight into the mechanisms leading to the synaptic alterations found in AD brain. Here, we discuss the implications of the oxidation of dihydropyrimidinase-related protein-2, glial fibrillary acid protein, and h-actin relative to the pathological changes noted in AD. Additionally, we discuss the implication of proteins that exhibited a trend toward increased oxidation and their relevance to AD.

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HPLC and MS verification of purity. The peptides were stored in the dry state at 20 -C until use. The oxyblot protein oxidation detection kit was purchased from Chemicon International (Temecula, CA, USA). 2.2. Preparation of synaptosomal membranes Synaptosomes were isolated from 3-month old male Mongolian gerbils. These animals were used since these rodent synaptosomes have been extensively characterized [63] and employed in neuroscience [31,34,40]. The procedure for isolating synaptosomes has been described elsewhere [34,63,68]. Briefly, the brain was immediately isolated and dissected following sacrifice by decapitation. The cortex was placed in 0.32 M sucrose isolation buffer containing 4 Ag/ml leupeptin, 4 Ag/ml pepstatin, 5 Ag/ml aprotinin (ICN Biomedicals, Aurora, OH, USA), 2 mM ethylenediaminetetraacetic acid (EDTA), 2 mM ethylene glycol-bistetraacetic acid (EGTA), 20 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES), 20 Ag/ ml trypsin inhibitor, and 0.2 mM phenylmethanesulfonyl fluoride (PMSF), pH 7.4. The tissue was homogenized by 20 passes with a Wheaton tissue homogenizer. The homogenate was centrifuged at 1500 g for 10 min. The supernatant was retained and centrifuged at 20,000 g for 10 min. The resulting pellet was resuspended in ¨1 ml of 0.32 M sucrose isolation buffer and layered over discontinuous sucrose gradients (0.85 M pH 8.0, 1.0 M pH 8.0, 1.18 M pH 8.5 sucrose solutions each containing 2 mM EDTA, 2 mM EGTA, and 10 mM HEPES) and spun at 82,500 g for 1 h at 4 -C. Synaptosomes were collected from the 1.0/ 1.18 M sucrose interface and washed in Locke’s buffer, pH 7.4 (154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 1.0 mM MgCl2, 3.6 mM NaHCO3, 5 mM glucose, 5 mM HEPES) twice for 10 min at 32,000 g. The resulting synaptosomal membranes were assayed for protein concentration by the Pierce BCA method. 2.3. Synaptosomal incubation with amyloid beta peptide Amyloid beta peptide 1 –42 or the non-oxidative and non-neurotoxic reverse peptide Ah(42 –1) [8,9,14] were solubilized in phosphate-buffered saline (PBS) to a final concentration of 1 mg/2 ml and preincubated for 24 h at 37 -C prior to incubation with synaptosomes. Synaptosomal preparations were incubated in 1 mg aliquots with or without 10 AM Ah(1– 42) in Locke’s Buffer for 6 h at 37 -C.

2. Materials and methods 2.4. Sample preparation 2.1. Chemicals All chemicals were of the highest purity and were obtained from Sigma (St. Louis, MO, USA) unless otherwise noted. Ah(1 –42) and amyloid h-peptide (42 – 1) were purchased from Anaspec (San Jose, CA, USA) with

According to the method of Levine et al. [42], synaptosomal samples were incubated at room temperature for 30 min in four volumes of 10 mM 2,4-dinitrophenylhydrazine (DNPH) in 2 M HCl for protein carbonyl derivatization/oxyblots or 2 M HCl for gel maps and mass

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spectrometry analysis. Proteins were precipitated by addition of ice-cold 100% trichloroacetic acid (TCA) to a final concentration of 15% for 10 min on ice. Precipitates were centrifuged for 2 min at 14,000 g 4 -C. The pellet was retained and washed with 500 Al of 1:1 (v/v) ethyl acetate/ ethanol three times. The final pellet was dissolved in rehydration buffer containing 8 M urea, 2 M thiourea, 2% CHAPS, 0.2% (v/v) biolytes, 50 mM dithiothreitol (DTT), and bromophenol blue. Samples were sonicated in rehydration buffer on ice three times for 20 s intervals. 2.5. Two-dimensional gel electrophoresis and Western blotting Two-dimensional polyacrylamide gel electrophoresis was performed with a Bio-Rad system using 110-mm pH 3 – 10 immobilized pH gradients (IPG) strips and Criterion 10% resolving gels. IPG strips were actively rehydrated at 50 V 20 -C overnight. 300 Ag of protein was loaded per strip by cup loading at the anode. Isoelectric focusing was performed at 20 -C as follows: 800 V for 2 h linear gradient, 1200 V for 4 h slow gradient, 8000 V for 8 h linear gradient, and 8000 V for 10 h rapid gradient. Gel strips were equilibrated for 10 min prior to second-dimension separation in 0.375 M Tris – HCl pH 8.8 containing 6 M urea (BioRad, Hercules, CA, USA), 2% (w/v) sodium dodecyl sulfate (SDS), 20% (v/v) glycerol, and 0.5% DTT (Bio-Rad, Hercules, CA, USA) followed by re-equilibration for 10 min in the same buffer containing 4.5% iodoacetamide (IA) (Bio-Rad, Hercules, CA, USA) in place of DTT. Control and Ah strips were placed on the Criterion gels, unstained molecular standards were applied, and electrophoresis was performed at 200 V for 65 min. 2.6. SYPRO Ruby staining Gels were fixed in a solution containing 10% (v/v) methanol, 7% (v/v) acetic acid for 20 min and stained overnight at room temperature with agitation in 50 ml of SYPRO Ruby gel stain (Bio-Rad, Hercules, CA, USA). 2.7. Oxyblot immunochemical detection For immunoblotting analysis, electrophoresis was performed as stated previously, and gels were transferred to a nitrocellulose membrane. The membranes were blocked with 3% bovine serum albumin (BSA) in phosphatebuffered saline containing 0.01% (w/v) sodium azide and 0.2% (v/v) Tween 20 (PBST) overnight at 4 -C. The membranes were incubated with anti-2,4-dinitrophenylhydrazone (DNP) polyclonal antibody diluted 1:100 in PBST for 2 h at room temperature with rocking. Following completion of the primary antibody incubation, the membranes were washed three times in PBST for 5 min each. An anti-rabbit IgG alkaline phosphatase secondary antibody was diluted 1:3000 in PBST and incubated with the

membranes for 2 h at room temperature. The membranes were washed in PBST three times for 5 min and developed using Sigmafast Tablets (BCIP/NBT substrate). Blots were dried and scanned with Adobe Photoshop. 2.8. In-gel digestion Samples were prepared according to the method described by Thongboonkerd et al. [61]. Briefly, the protein spots were cut and removed from the gel with a clean razor blade. The gel pieces were placed into individual, clean 1.5 ml microcentrifuge tubes, and kept overnight at 20 -C. The gel pieces were thawed and washed with 0.1 M ammonium bicarbonate (NH4HCO3) for 15 min at room temperature. Acetonitrile was added to the gel pieces and incubated for an additional 15 min. The liquid was removed, and the gel pieces were allowed to dry. The gel pieces were rehydrated with 20 mM DTT (Bio-Rad, Hercules, CA, USA) in 0.1 M NH4HCO3 and incubated for 45 min at 56 -C. The DTT was removed and replaced with 55 mM IA (Bio-Rad, Hercules, CA, USA) in 0.1 M NH4HCO3 for 30 min in the dark at room temperature. The liquid was drawn off, and the gel pieces were incubated with 50 mM NH4HCO3 at room temperature for 15 min. Acetonitrile was added to the gel pieces for 15 min at room temperature. All solvents were removed, and the gel pieces were allowed to dry for 30 min. The gel pieces were rehydrated with addition of a minimal volume of 20 ng/Al modified trypsin in 50 mM NH4HCO3. The gel pieces were chopped and incubated with shaking overnight (¨18 h) at 37 -C. 2.9. Analysis of gel images The analysis of the gel maps and membranes to compare protein expression and carbonyl immunoreactivity content between control and Ah treated samples was performed with PDQuest software (Bio-Rad, Hercules, CA, USA). Images from SYPRO Ruby stained gels, used to measure protein content, were obtained using a UV transilluminator (k ex = 470 nm, k em = 618 nm, Molecular Dynamics, Sunnyvale, CA, USA). Oxyblots, used to measure carbonyl immunoreactivity, were scanned with a Microtek Scanmaker 4900. 2.10. Mass spectrometry For this study, all mass spectra were recorded at the University of Kentucky Mass Spectrometry Facility (UKMSF). A Bruker Autoflex MALDI-TOF (matrix assisted laser desorption-time of flight) mass spectrometer (Bruker Daltonic, Billerica, MA, USA) operated in the reflection mode was used to generate peptide mass fingerprints. Peptides resulting form in-gel digestion were analyzed on a 384 position, 600 Am Anchor-Chipi Target (Bruker Daltonics, Bremen, Germany) and prepared according to AnchorChip recommendations (AnchorChip Tech-


nology, Rev. 2, Bruker Daltonics, Bremen Germany). Briefly, 1 Al of digestate was mixed with 1 Al of alphacyano-4-hydroxycinnamic acid (0.3 mg/ml in ethanol:acetone, 2:1 ratio) directly on the target and allowed to dry at room temperature. The sample spot was washed with 1 Al of 1% TFA solution for approximately 60 s. The TFA droplet was gently blown off the sample spot with compressed air. The resulting diffuse sample spot was recrystallized (refocused) using 1 Al of a solution of ethanol:acetone:0.1% TFA (6:3:1 ratio). Reported spectra are a summation of 100 laser shots. External calibration of the mass axis was used for acquisition, and internal calibration using either trypsin autolysis ions or matrix clusters was applied post acquisition for accurate mass determination. LC/MS/MS spectra were acquired on a Finnigan LCQ FClassic_ quadrupole ion trap mass spectrometer (Finnigan, Co., San Jose, CA, USA). Separations were performed with an HP 1100 HPLC modified with a custom splitter to deliver 4 Al/min to a custom C18 capillary column (300 Am id 15 cm, packed in-house with Macrophere 300 5 Am C18) (Alltech Associates, Deerfield, IL, USA). Gradient separations consisted of 2 min isocratic at 95% water: 5% acetonitrile (both phases contain 0.1% formic acid), the organic phase was increased to 20% acetonitrile over 8 min, then increased to 90% acetonitrile over 25 min, held at 90% acetonitrile for 8 min, then increased to 95% in 2 min, and finally returned to initial conditions in 10 min (total acquisition time 45 min with a 10-min recycle time). Tandem mass spectra were acquired in a data-dependent manner. Three microscans were averaged to generate the data-dependent full scan spectrum. The most intense ion was subjected to tandem mass spectrometry, and three microscans were averaged to produce the MS/MS spectrum. Mass subjected to the MS/MS scan was placed on an exclusion list for 2 min. 2.11. Analysis of peptide sequences Peptide mass fingerprinting was used to identify proteins from tryptic peptide fragments by utilizing the MASCOT search engine (http://www.matrixscience.com) based on the entire NCBI and SwissProt protein databases. Database searches were conducted allowing for up to one missed trypsin cleavage and using the assumption that the peptides were monoisotopic, oxidized at methionine residues, and carbamidomethylated at cysteine residues. Mass tolerance of 150 ppm/g was the window of error allowed for matching the peptide mass values. Probability-based MOWSE scores were estimated by comparison of search results against estimated random match population and were reported as 10 * Log10( p), where p is the probability that the identification of the protein is not correct. MOWSE scores greater than 63 were considered to be significant ( P < 0.05). All protein identifications were in the expected size and pI range based on position in the gel.

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2.12. Statistical analysis Statistical comparison of carbonyl levels of proteins, matched with anti-DNP positive spots on 2D-oxyblots from synaptosomal samples treated with Ah(1 –42) and untreated control synaptosomal samples, was performed using ANOVA. P values of < 0.05 were considered to be significant.

3. Results Comparison of synaptosomal protein oxidation levels in synaptosomes treated with Ah(1 –42) and control synaptosomal samples was carried out by first identifying carbonylated proteins via anti-DNP immunochemical development of proteins transferred to a nitrocellulose membrane or 2Doyxblot analysis (Fig. 1B). Individual protein spots were matched between the 2D-PAGE maps and the 2D-oxyblots, and the carbonyl immunoreactivity of each spot was normalized to the protein content in the 2D-PAGE (Fig. 1A). In this study, we confirm previous reports that many, but not all, individual proteins exhibit carbonyl immunoreactivity [19 – 22]. Mass spectrometry analysis allowed for the identification of protein spots found to be increasingly carbonylated by Ah(1 –42). Dihydropyrimidinase-related protein-2 (DRP2), glial fibrillary acidic protein (GFAP), and h-actin were found exhibit a significant increase in protein carbonylation. Additionally, g-actin, H+-transporting two-sector ATPase, syntaxin binding protein 1, glutamate dehydrogenase, and elongation factor Tu were found to exhibit a trend toward increased carbonylation by Ah(1 – 42). These findings are summarized in Fig. 2. Using MASCOT, the probability-based MOWSE score was 288 for DRP-2, with 6 peptide matches and 14% sequence coverage, 72 for hactin, with 8/29 peptide matches and 29% sequence coverage, 61 for GFAP, with 7/26 peptide matches and 12% sequence coverage, 107 for g-actin, with 17/52 peptide matches and 44% sequence coverage, 69 for H+transporting two-sector ATPase, with 8/28 peptide matches and 20% sequence coverage, 106 for syntaxin binding protein 1, with 13/28 peptide matches and 20% sequence coverage, 84 for glutamate dehydrogenase, with 10/29 peptide matches and 16% sequence coverage, and 111 for elongation factor Tu, with 4 peptide matches and 8% sequence coverage. The increase in carbonylation compared to control was significant for DRP-2 (341 T 65% control, P < 0.04), GFAP (7426 T 622% control, P < 0.04), and hactin (498 T 93% control, P < 0.04). A non-significant increase in protein oxidation was detected for g-actin (335 T 101% control, P > 0.05), H+-transporting two-sector ATPase (13490 T 4725% control, P > 0.05), syntaxin binding protein 1 (739 T 120% control, P > 0.05), glutamate dehydrogenase (2360 T 446% control, P > 0.05), and elongation factor Tu mitochondrial form (206 T 41% control, P > 0.05). Based on

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Fig. 1. SYPRO Ruby stained 2D gels (A) and 2D-oxyblots (B) from control synaptosomes and synaptosomes treated with Ah(1 – 42). The boxes outline the area enlarged in Fig. 2.

the MOWSE score, the identification of GFAP is not significant, however, the molecular weight and pI of the protein spot of interest corresponded with that of GFAP. Similar studies with the non-oxidative and non-neurotoxic, reverse peptide Ah(42 –1) [8,9,14] revealed a pattern of oxidized proteins similar to that seen in untreated control synaptosomes (data not shown), demonstrating that our reported results reflect Ah(1– 42)-induced protein oxidation and not artifactual effects of any peptide addition to synaptosomes. Information about the proteins identified in this study is summarized in Table 1.

4. Discussion Protein oxidation can lead to a variety of cellular consequences including decreased protein turnover, increased protein aggregation, loss of protein function, altered cellular redox potential, mitochondrial dysfunction, and ultimately cell death [52]. There is increasing evidence that protein oxidation plays a role in AD [15]. Additionally, there is increasing evidence that Ah(1 –42) may play a central role in the pathogenesis of AD as a mediator of oxidative stress [14]. Synapse loss is believed to be an early

Fig. 2. Enlargements of 2D gel (A) and 2D oxyblot (B) images show the position of protein spots and carbonyl immunoreactivity, respectively. The 2D-oxyblot from Ah(1 – 42) treated synaptosomes is labeled with the proteins identified in this study.


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Table 1 Summary of the proteins identified to be increasingly carbonylated in synaptosomes treated with Ah(1 – 42)a Protein

MOWSE score

Peptides matched

% Coverage

% Oxidation

Gamma-actin Beta-actin Glial fibrillary acidic protein H+-transporting two-sector ATPase Syntaxin binding protein 1 Glutamate dehydrogenase Dihydropyrimidinase-related protein-2 Elongation factor Tu (mt)

107 72 61b 69 106 84 288 111

17 8 7 8 13 10 6 4

44 29 12 20 20 16 14 8

335 498 7426 13,490 739 2360 341 206

T T T T T T T T

152 164 3787 9401 483 1787 80 91

MW

pI

P value

41,335 39,446 49,887 51,171 68,058 61,640 62,277 49,508

5.56 5.78 5.41 4.92 6.62 8.05 5.95 7.23