Molecular Brain Research 138 (2005) 8 – 16 www.elsevier.com/locate/molbrainres Research Report Proteomic identification of less oxidized brain proteins in aged senescence-accelerated mice following administration of antisense oligonucleotide directed at the Ah region of amyloid precursor protein H. Fai Poona, Susan A. Farrb,c, William A. Banksb,c, William M. Pierced, Jon B. Kleine, John E. Morleyb,c, D. Allan Butterfielda,* a Department of Chemistry, Center of Membrane Sciences, and Sander-Brown Center on Aging, University of Kentucky, Lexington, KY 40506-0055, USA b Geriatric Research Education and Clinical Center (GRECC), VA Medical Center, St. Louis, MO 63106, USA c Department of Internal Medicine, Division of Geriatric Medicine, St. Louis University School of Medicine, St. Louis, MO 63110-0250, USA d Department of Pharmacology, University of Louisville School of Medicine and VAMC, Louisville, KY 40292, USA e Kidney Disease Program and Proteomics Core Laboratory, University of Louisville School of Medicine and VAMC, Louisville, KY 40292, USA Accepted 13 February 2005 Available online 1 June 2005 Abstract Amyloid h-peptide (Ah) is the major constituent of senile plaques, a pathological hallmark of Alzheimer’s disease (AD) brain. It is generally accepted that Ah plays a central role in the pathophysiology of AD. Ah is released from cells under entirely normal cellular conditions during the internalization and endosomal processing of amyloid precursor protein (APP). However, accumulation of Ah can induce neurotoxicity. Our previous reports showed that decreasing the production of Ah by giving an intracerebroventricular injection of a 42-mer phosphorothiolated antisense oligonucleotide (AO) directed at the Ah region of the APP gene reduces lipid peroxidation and protein oxidation and improves cognitive deficits in aged senescence-accelerated mice prone 8 (SAMP8) mice. In order to investigate how Ah level reduction improves learning and memory performance of SAMP8 mice through reduction of oxidative stress in brains, we used proteomics to identify the proteins that are less oxidized in 12-month-old SAMP8 mice brains treated with AO against the Ah region of APP (12 mA) compared to that of the age-control SAMP8 mice. We found that the specific protein carbonyl levels of aldoase 3 (Aldo3), coronin 1a (Coro1a) and peroxiredoxin 2 (Prdx2) are significantly decreased in the brains of 12 mA SAMP8 mice compared to the age-controlled SAMP8 treated with random AO (12 mR). We also found that the expression level of a-ATP synthase (Atp5a1) was significantly decreased, whereas the expression of profilin 2 (Pro-2) was significantly increased in brains from 12 mA SAMP8 mice. Our results suggest that decreasing Ah levels in aged brain in aged accelerated mice may contribute to the mechanism of restoring the learning and memory improvement in aged SAMP8 mice and may provide insight into the role of Ah in the memory and cognitive deficits in AD. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Degenerative disease: Alzheimer’s beta amyloid Keywords: Proteomic identification; SAMP8; Amyloid precursor protein; Antisense oligonucleotide; Amyloid beta-peptide; Alzheimer’s disease; Oxidative stress 1. Introduction disease (AD) [6,25]. Senile plaques contain extracellular deposits of amyloid-h peptides (Ah) in star-shaped masses Senile plaques, neurofibrillary tangles (NFTs) and of amyloid fibrils, and NFTs are composed of the micro- synapse loss are three pathological features of Alzheimer’s tubule-associated protein tau [7,42,51,63,85]. There is growing evidence that the formation of NFTs in AD is * Corresponding author. Fax: +1 859 257 5876. one of the neuronal responses to the accumulation of Ah E-mail address: email@example.com (D.A. Butterfield). and Ah-associated stress . Ah is released from cells 0169-328X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2005.02.020 H.F. Poon et al. / Molecular Brain Research 138 (2005) 8 – 16 9 under entirely normal cellular conditions [10,43,72,73] 2. Materials and methods during the internalization and endosomal processing of APP [50,65]. However, accumulation of Ah can induce All chemicals were purchased from Sigma-Aldrich neurotoxicity by binding to the nicotinic acetylcholine unless otherwise specified. receptor , forming calcium and potassium channels in cell membranes [3,26,27], decreasing glucose transport 2.1. Subjects across brain endothelial cells  and actuating the release of chemokines  and cytokines . Moreover, Ah can The SAMP8 mice were from an in-house colony, inbred induce oxidative damage directly to cells through its ability for 10 years from stock obtained from Dr. Takeda of Kyoto to produce free radicals [11– 13,15,16,81]. Ah-mediated University, Japan. Mice were on a 12 h light:12 h dark cycle S oxidative stress also includes the production of O2 À through with lights on at 06:00. Food and water were available ad the stimulation of NADPH oxidase ; the production of libitum. Sentinels from the colony have remained free of H2O2 through copper or iron reduction; and the production pathogens including mycoplasma, salmonella/shigella, ecto- of nitric oxide (NO) in macrophages in a microglial cell line parasites, pneumonia virus, Sendai virus, mouse hepatitis, . These Ah-mediated reactive oxygen species (ROS) Reo 3, ectromelia, GBVII and lymphcytic choriomeningitis. can further oxidatively modify proteins, lipids and DNA to elicit damage in cells. 2.2. Antisense administration When Ah is administered exogenously, it causes deficits in learning and memory in animals [21,23,34,36,74,77,86]. Phosphorothiolated antisense oligonucleotides (AO) were Such learning and memory impairments are also observed in designed to be directed at positions 17 to 30 of the Ah region overexpressed Ah transgenic models  and the senes- of the APP gene (Midland Certified Reagent Company, cence-accelerated mice prone 8 (SAMP8) mice [28,31 – Midland, TX). The sequences of the designated AO and of a 33,35,37,62]. Aged SAMP8 mice show impaired age- random AO used as a control were as previously described dependent defects of learning and memory , along with [53,68]. These AOs were previously used to reverse the Ah-like-protein immunoreactive granular structures in their learning and memory deficit and reduce oxidative stress in central nervous system (CNS) . Brains of SAMP8 mice aged SAMP8 mouse [53,68]. Administration of AO was also show spheroidal axonal dystrophy in dorsal column performed according to Kumar et. al. . Mice were nuclei, small neurons in gracile nucleus and some well- anesthetized in a stereotactic instrument with methoxyflur- defined or swollen axons . Age-related shrinkage of the ane. Three injections of vehicle or AO were given to the mice cholinergic neurons of the laterodorsal tegmental nucleus by intracerebroventricular (ICV) injection on 2 weeks are observed in aged SAMP8 mice brains , indicating intervals (from 11 months to 12 months). All substances abnormal axonal transport, synaptic terminal remodeling (65 ng) were injected in a 2 AL volume by drilling a hole and/or actin polymerization/depolymerization occur in through the skull over the third ventricle (À0.5 relative to SAMP8 mice brains. Bregma; 0.5 mm right of central suture). The scalp wound SAMP8 mice show increased oxidative stress in their was closed and the mice were returned to their cages. CNS as a function of age [14,28,67]. With treatments of antioxidants, such as a-lipoic acid, N-tert-butyl-alpha- 2.3. Sample preparation and methods employed phenylnitrone, carnitine, acetyl-l-carnitine and melatonin, the lipid peroxidation and protein oxidation in SAMP8 mice Mice were sacrificed 2 weeks after the last injection. were reduced [14,28,64,87]. Some of these antioxidants can SAMP8 mouse whole brains were flash frozen in liquid also reverse the learning and memory impairments observed nitrogen. The whole brain samples were homogenized in a in aged SAMP8 mice [28,87]. Similar to other antioxidant lysis buffer (pH 7.4, 10 mm HEPES, 137 mm NaCl, 4.6 mm studies, our previous reports showed that decreasing the KCl, 1.1 mm KH2PO4, 0.6 mm MgSO4 and 0.5 mg/mL production of Ah by giving an intracerebroventricular leupeptin, 0.7 Ag/mL pepstatin (ICN Biomedicals, Irvine, injection of a 42-mer phosphorothiolated antisense oligo- CA), 0.5 Ag/mL trypsin inhibitor and 40 Ag/mL PMSF (ICN nucleotide (AO) directed at the Ah region of the APP gene Biomedicals, Irvine, CA)). Homogenates were centrifuged can reduce lipid peroxidation and protein oxidation  and at 15,800 Â g for 10 min to remove debris. The supernatant improve cognitive deficits  in aged SAMP8 mice. In was extracted to determine the concentration by the BCA order to investigate how Ah level reduction improves method (Pierce, Rockford, IL). learning and memory performance of SAMP8 mice through reduction of oxidative stress in brains, we used proteomics 2.4. Two-dimensional electrophoresis to identify the proteins that are less oxidized, as well as the proteins that are expressed differently, in 12-month-old Samples of brain proteins were prepared according to the SAMP8 mice brains treated with AO against the Ah region procedure of Levine et al. . Proteins (200 Ag) were of APP (12 mA) compared to brain from age-controlled incubated with 4 volumes of 2 N HCl at room temperature SAMP8 mice treated with random AO (12 mR). (25 -C) for 20 min. Proteins were then precipitated by 10 H.F. Poon et al. / Molecular Brain Research 138 (2005) 8 – 16 addition of ice-cold 100% trichloroacetic acid (TCA) to stain was developed by application of Sigma-Fast (BCIP/ obtain a final concentration of 15% TCA. Samples are NBT) tablets. placed on ice for 10 min to let proteins precipitate. Precipitates were centrifuged at 15,800 Â g for 2 min. This 2.6. Image analysis process removed ions that affect the voltage during the isoelectric focusing. The pellets were washed with 1 mL of The gels and nitrocellulose papers were scanned and 1:1 (v/v) ethanol/ethyl acetate solution. After centrifugation saved in TIF format using Strom 860 Scan (Molecular and washing with ethanol/ethyl acetate solution three times, Dynamics, Sunnyvale, CA) and Scanjet 3300 C (Hewlett the samples were dissolved in 25 AL of 8 M urea (Bio-Rad, Packard), respectively. PDQuest 2D analysis software (Bio- Hercules, CA). The samples then were mixed with 185 AL Rad, Hercules, CA) was used for matching and analysis of of rehydration buffer (8 M urea (Bio-Rad, Hercules, CA), 20 visualized protein spots among differential gels and oxy- mM dithiothreitol (Bio-Rad, Hercules, CA), 2.0% (w/v) blots. The principles of measuring intensity values by 2D CHAPS, 0.2% Biolytes (Bio-Rad, Hercules, CA), 2 M analysis software were similar to those of densitometric thiourea and bromophenol blue). measurement. Average mode of background subtraction was In first-dimension electrophoresis, 200 AL of sample used to normalize intensity value, which represents the solution were applied to a ReadyStripi linear IPG strip (11 amount of protein (total protein on gel and oxidized protein cm, Bio-Rad, Hercules, CA). The strips were soaked in the on oxyblot per spot). After completion of spot matching, the sample solution for 1 h to ensure uptake of the proteins. The normalized intensity of each protein spot from individual strip was then actively rehydrated in the protean IEF cell gels (or oxyblots) was compared between the aged SAMP8 (Bio-Rad, Hercules, CA) for 16 h at 50 V. The isoelectric mice (n = 7) and the aged SAMP8 mice treated with AO focusing was performed at 300 V for 2 h linearly; 500 V for (n = 7) using Student’s t test statistical analysis. 2 h linearly; 1000 V for 2 h linearly, 8000 V for 8 h linearly and 8000 V for 10 h rapidly. All the processes above were 2.7. Trypsin digestion carried out at 22 -C. The strip was stored in À80 -C until the second dimension electrophoresis was performed. Samples were prepared using techniques described by For the second dimension, IPG\ Strips (Bio-Rad, Jensen , modified by Thongboonkerd et al. . The Hercules, CA) pH 3– 10 were equilibrated for 10 min in protein spots were excised with a clean blade and transferred 50 mm Tris –HCl (pH 6.8) containing 6 m urea, 1% (w/v) into clean microcentrifuge tubes. The protein spots were then sodium dodecyl sulfate (SDS), 30% (v/v) glycerol and 0.5% washed with 0.1 M ammonium bicarbonate (NH4HCO3) at dithiothreitol and then re-equilibrated for 15 min in the same room temperature for 15 min. Acetonitrile was added to the buffer containing 4.5% iodoacetamide in place of dithio- gel pieces and incubated at room temperature for 15 min. threitol. Linear gradient 8– 16% precast criterion Tris –HCl The solvent was removed, and the gel pieces were dried in a gels (13 cm Â 8 cm Â 1 mm, Bio-Rad, Hercules, CA) were flow hood. The protein spots were incubated with 20 AL of used to perform second dimension electrophoresis. Preci- 20 mM DTT in 0.1 M NH4HCO3 at 56 -C for 45 min. The sion Proteini Standards (Bio-Rad, Hercules, CA) were run DTT solution was then removed and replaced with 20 AL of along with the sample at 200 V for 65 min. 55 mM iodoacetamide in 0.1 M NH4HCO3. The solution was The gel was incubated in fixing solution (7% acetic acid, incubated at room temperature in the dark for 30 min. The 40% methanol) for 20 min after the second dimension iodoacetamide was removed and replaced with 0.2 mL of 50 electrophoresis. Approximately, 60 mL of Sypro Ruby stain mM NH4HCO3 and incubated at room temperature for 15 (Bio-Rad, Hercules, CA) were used to stain the gel for 2 h. min. 200 AL of acetonitrile was added. After 15 min The gels were destained in deionized water overnight. incubation, the solvent was removed, and the gel spots were dried in a flow hood for 30 min. The gel pieces were 2.5. Western blotting rehydrated with 20 ng/AL modified trypsin (Promega, Madison, WI) in 50 mM NH4HCO3 with the minimal Proteins (200Ag) were incubated with 4 volumes of 20 volume to cover the gel pieces. The gel pieces were chopped mM DNPH at room temperature (25 -C) for 20 min. The into smaller pieces and incubated at 37 -C overnight in a gels were prepared in the same manner as for 2D-electro- shaking incubator. phoresis. After the second dimension, the proteins from gels were transferred to nitrocellulose papers (Bio-Rad, Her- 2.8. Mass spectrometry cules, CA) using the Transblot-Blot\ SD semi-Dry Transfer Cell (Bio-Rad, Hercules, CA) at 15 V for 2 h. The 2,4- Digests (1 AL) were mixed with 1 AL a-cyano-4-hydroxy- dinitrophenyl hydrazone (DNP) adduct of the carbonyls of trans-cinnamic acid (10 mg/mL in 0.1% TFA:ACN, 1:1, the proteins was detected on the nitrocellulose paper using a v/v). The mixture (1 AL) was deposited onto a fast primary rabbit antibody (Chemicon, Temecula, CA) specific evaporation nitrocellulose matrix surface, washed twice for DNP – protein adducts (1:100), and then a secondary with 2 AL 5% formic acid and analyzed with a TofSpec 2 goat anti-rabbit IgG antibody was applied. The resultant E (Micromass, UK) MALDI-TOF mass spectrometer in H.F. Poon et al. / Molecular Brain Research 138 (2005) 8 – 16 11 Table 1 Summary of proteins identified by mass spectrometry Identified proteins Databse accession # Peptide matches % Coverage matched pI, MrW Mowse code identified peptides Score Profilin 2 PRO2_ MOUSE 6 38 6.78, 14.9 68 ATP synthase alpha subunit, isoform 1 gi|6680748 14 26 9.22, 59.7 123 Aldo3 protein gi|13435924 8 25 6.47, 39.3 87 Peroxiredoxin 2 Q9CWJ4 7 34 5.70, 21.8 74 Coronin, actin binding protein 1A gi|31418362 10 16 6.05, 51.0 94 Mowse scores greater than 62 are considered significant (P < 0.05). reflectron mode. The mass axis was adjusted with trypsin 3. Results autohydrolysis peaks (m/z 2239.14, 2211.10, or 842.51) as lock masses. The specific carbonyl level, obtained by dividing the The MALDI spectra used for protein identification from carbonyl level of a protein spot on the nitrocellulose tryptic fragments were searched against the MSDB protein membrane by the protein level of its corresponding protein databases using the MASCOT search engine (http:// spot on the gel, indicates the carbonyl level per unit of www.matrixscience.com). Peptide mass fingerprinting used protein. 144 proteins on 2D gels and 2D Western blots the assumption that peptides are monoisotopic, oxidized at were matched between 12 mR SAMP8 and 12 mA methionine residues and carbamidomethylated at cysteine SAMP8. In comparison to 12 mR SAMP8 mice, 12 mA residues [17 –20]. Up to 1 missed trypsin cleavage was SAMP8 mice brains have two proteins that are expressed allowed. Mass tolerance of less than 100 ppm was the differently in terms of protein levels and three proteins that window of error allowed for matching the peptide mass have significantly lower specific carbonyl levels. The values. The probability-based Mowse score is assigned for identified proteins are listed in Table 1. All the mass each spectrum to indicate the probability that the match spectra (not shown) of the peptides were matched to the between the database and the spectra is a random event. mass spectra in MSDB protein databases with reported Scores greater than 62 were considered significant. Thus, if probability-based Mowse score. All protein identifications a match has a score higher than 62, the match as a random agree with the expected MrW and pI range based on their event is not significant. positions on the gel. Fig. 1. (12 mR) Representative gels of proteins after 2D electrophoresis from the brain of 12-month-old SAMP8 mice injected with random AO. (12 mA) Proteins after 2D electrophoresis from the brain of 12-month-old SAMP8 mice treated with AO directed to the Ah region of APP. Insets: (right) expanded image of regions of the 2D gel of SAMP8 mice brains outlined in the box. 12 H.F. Poon et al. / Molecular Brain Research 138 (2005) 8 – 16 Table 2 Table 3 Proteins expressed differently when 12-month-old SAMP8 mice are Proteins that have specific carbonyl level decreased by AO injected with AO compared to non-treated 12-month-old SAMP8 mice Identified proteins Specific protein carbonyl P value Identified proteins Protein levels of P value levels of 12-month-old 12-month-old SAMP8 SAMP8 injected with AOa injected with AOa Aldo 3 9.4 T 0.3 <0.000001 Alpha-ATP synthase 54.5 T 11.2 <0.05 Coronin1A 46.4 T 16.3 <0.005 Profilin 2 139 T 7.8 <0.05 Peroxiredoxin 2 26.8 T 12 <0.005 a a Percent of the level found in brain from 12 mR-treated SAMP8 mice. Percent of the level found in brain from 12 mR-treated SAMP8 mice. coronin 1a (Coro1a) and peroxiredoxin 2 (Prdx2) were 3.1. Protein expression levels significantly decreased in the brain of 12 mA SAMP8. Fig. 1 shows the representative gel of the whole brain homogenate from 12 mR and 12 mA-treated SAMP8 mice. 4. Discussion The proteins that were expressed differently in SAMP8 brains are summarized in Table 2. The expression level of a- We previously showed that AO was successfully ATP synthase (Atp5a1) was significantly decreased, internalized by neurons and reduced the level of Ah, thus whereas the expression of profilin 2 (Pro-2) was signifi- reducing Ah-mediated protein oxidation and lipid perox- cantly increased in brain from 12-month-old SAMP8 mice idation in 12 mA SAMP8 mice brains . Here, we show treated with AO. that the specific carbonyl levels of Aldo3, Coro2 and Prdx2 in the 12 mA SAMP8 mice brains are significantly 3.2. Specific protein carbonyl level decreased when compared to those in the 12 mR SAMP8 mice. We also find that the protein levels of Pro-2 and Fig. 2 shows the representative Western blots of 12 mR Atp5a1 in brains of 12 mA SAMP8 mice are significantly and 12 mA SAMP8 mice brains. The summary of the altered when compared to 12 mR SAMP8 mice brains. In specific protein carbonyl levels is given in Table 3. The our previous study, we found that the specific carbonyl level specific protein carbonyl levels of aldoase 3(Aldo3), of Aldo3, Coro1a and Prdx2 were increased (486%, 599% Fig. 2. (12 mR) Carbonyl Western blot from the brain of 12-month-old SAMP8 mice injected with random AO (left) and expansion of the blot outlined in the box (right). (12 mA) Carbonyl Western blot from the brain of 12-month-old SAMP8 mice treated with AO directed to Ah region of APP (left) and expansion of the blot outlined in the box (right). H.F. Poon et al. / Molecular Brain Research 138 (2005) 8 – 16 13 and 223%, respectively) in 12-month-old SAMP8 mice metabolic pathway, increase ATP production for antioxidant brains when comparing to that in the 4-month-old SAMP8 defensive systems and synaptic elements in neurons of mice brains (unpublished). However, these increases of the SAMP8 mice brains. specific carbonyl levels are not statistically significant, thus Prdx2 is an antioxidant enzyme that is exclusively were not reported in the previous paper . expressed in neurons . Since Prdx2 modulates intra- Coro1a is an actin binding protein  that promotes cellular H2O2 production and H2O2 mediated apoptosis rapid actin polymerization by reducing the lag phase of actin , it is possible that Prdx2 is responsible for the polymerization. The coro1a level in fetal Down’s syndrome elimination of Ah-mediated H2O2 production in neurons. (DS) brain is reduced, indicating that it plays a role in The increased expression of Prdx2 in AD, Down’s migration of cells and/or neuronal outgrowth . There- syndrome and PD [49,52] is likely to be in response to fore, oxidative inactivation of Coro1a might contribute to the increased oxidative stress and/or its activity loss due to the neuronal shrinkage observed in SAMP8 mice brains, oxidative modification. Therefore, reducing Ah level in thus leading to learning and memory impairment similar to SAMP8 mice brain by i.c.v. injection of AO can reduce the DS patients. Here, we show that the specific carbonyl level H2O2 production and therefore reduce the specific carbonyl of Coro1a was reduced with the treatment of AO to reduce level of Prdx2, suggesting that more active Prdx2 is Ah levels in SAMP8 mice brains, suggesting the oxidative available in neurons to strengthen its antioxidant system. inactivation of Coro1a was possibly recovered. Thus, the This may result in improving the biological function of normal neuronal outgrowth dynamic and neuronal functions neurons in aged SAMP8 mice and contribute to the presumably were reestablished by the AO treatment in improvement in learning and memory. SAMP8 mice brains. Pro-2, expressed in neuronal cells, is an isoform of the Aldo3 is a brain-specific glycolytic enzyme. Reduced profilin family. Profilin binds to monomeric actin to prevent activity of Aldo3 was reported in aged rats, suggesting that the abnormal polymerization of actin into filaments. Pro-2 oxidative modification may be responsible for Aldo3 also promotes actin polymerization . The expression of activity decline in aged animals . Moreover, Aldo3 profilin is regulated by oxidative stress instead of protein interacts with dihydropyrimidinase-like 2 (DRP-2) in the kinase C , and downregulation of profilin is also complex of NADH – dichorophen – indophenol (DCIP) observed in oxidative-stressed conditions, i.e. hypoxia reductase in response to the external oxidative stress and/ . Profilin is reported as a glutathionylated protein in or antioxidants . Here, we show that specific carbonyl oxidatively stressed cell lines [39,40], suggesting that level of Aldo3 was significantly reduced in 12 mA SAMP8 glutathionylation might be a common mechanism for the mice by i.c.v. injection of AO when compared to that in 12 global regulation of profilin function. Our current study mR mice brains, which should partially restore the impaired shows that the Pro-2 expression is increased in the brain of Fig. 3. Functional interacteome of proteins described in this study. Interacteome indicates that all of the proteins are directly and/or indirectly interacting with actin and actin polymerization, an important process for neuroplasticity. 14 H.F. Poon et al. / Molecular Brain Research 138 (2005) 8 – 16 SAMP8 mice treated with AO, suggesting that the neuronal Acknowledgment outgrowth dynamic by actin is restored, which in turn possibly secures neuronal junction and improves learning This work was supported in part by grant from NIH to and memory in SAMP8 mice. D.A.B [AG-05119; AG-10836]. Atp5a1 is a member of the F1 synthase enzymatic complex that binds ADP, phosphate and ATP for the synthesis of ATP during oxidative phosphorylation. Atp5a1 References is an inner mitochondrial membrane protein. However,  K.T. Akama, L.J. 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