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Analysis of brain proteins in Alzheimer’s disease using high-resolution two-dimensional gel electrophoresis

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Analysis of brain proteins in Alzheimer’s disease using high-resolution two-dimensional gel electrophoresis
  Journal of the Neurological Sciences 166 (1999) 100–106 Analysis of brain proteins in Alzheimer’s disease using high-resolutiontwo-dimensional gel electrophoresis a a, a b b *T. Tsuji , S. Shimohama , S. Kamiya , T. Sazuka , O. Ohara a  Department of Neurology ,  Faculty of Medicine ,  Kyoto University , 54   Shogoin - Kawaharacho ,  Sakyoku ,  Kyoto  606,  Japan b  Laboratory of DNA Technology ,  Kazusa DNA Research Institute , 1532  - 3   Yana ,  Kisarazu - shi ,  Chiba  292,  Japan Received 10 December 1998; received in revised form 6 May 1999; accepted 10 May 1999 Abstract Two-dimensional gel electrophoresis (2-DE), a method which can be used to analyze the expression of many proteins, is a promisingand powerful approach which we have begun to use in the characterization of the complex pathologic processes in Alzheimer’s disease(AD). In the present study, a reliable 2-DE database of human brain proteins was created by improving the reproducibility of 2-DE imagesusing an immobilized pH gradient (IPG) for the first dimension gel electrophoresis and Melanie II as the program for data analysis. Thebrain samples were taken from the temporal cortex of brains at autopsy from 15 AD patients and 15 age-matched controls withnon-neurological disorders. About 700 spots were located as consistently expressed proteins in the human brain, all of which wereexpressed also in AD brains. Comparing the density of spots between AD and normal control, we found that five protein spots weresignificantly increased, 28 spots were significantly decreased and nine spots were detected only in AD. Two spots among thosesignificantly increased and one spot among those significantly decreased were identified as glial fibrillary acidic proteins. The database of brain proteins in AD constructed for the present study, including the statistical data of density changes in AD, should be a usefulbeginning for a comprehensive human 2-DE database available via the Internet, which will facilitate further investigation of pathogenicprotein alterations in AD.  ©  1999 Elsevier Science B.V. All rights reserved. Keywords :   Two-dimensional gel electrophoresis; Alzheimer’s disease; Protein; Database; Internet 1. Introduction  associated with diseases [11], an ongoing process highlyimportant to AD research. Studies at the protein level,Recent research on Alzheimer’s disease (AD) has however, have lagged partly due to the complexity of yielded many fruitful and rapidly unfolding observations techniques required for separation, analysis, and identifica-relating to its pathogenesis. Various proteins such as tion.amyloid precursor protein (APP),  b -amyloid, tau, pre- Two-dimensional gel electrophoresis (2-DE) has beensenilin and apolipoprotein E are likely to be involved in developed as a method of protein separation combiningthe development of this disease [1–7]. Recent advances in isoelectric focusing gel electrophoresis (IEF) with sodiummolecular biology techniques have enabled us to identify dodecyl sulfate (SDS) polyacrylamide gel electrophoresiscandidate genes for familial AD [8–10]. Furthermore, the (PAGE). This combination can be used to separate andHuman Genome Project has brought about remarkable characterize many thousands of proteins detected as spotsadvances in both genetic maps and identification of genes on the gels or transferred membranes [12]. Measurement of changes in the expression of the multiple proteins providesa powerful strategy for characterizing complex *Corresponding author. Tel.:  1 81-75-751-3767; fax:  1 81-75-751- pathophysiologic processes and designing novel drug 9541.  E  - mail address :   i53367@sakura.kudpc.kyoto-u.ac.jp (S. Shimohama)  therapies. 0022-510X/99/$ – see front matter  ©  1999 Elsevier Science B.V. All rights reserved.PII: S0022-510X(99)00120-3  T  .  Tsuji et al .  /   Journal of the Neurological Sciences  166 (1999) 100  – 106   101 Despite the low number of proteins detected on 2-DE both groups the typical cause of death was cardiac failurecompared with the estimated 50 000–100 000 human or a terminal respiratory condition. Immediately aftergenes expressed in the adult brain, 2-DE analysis is autopsy the brains were divided sagittally into halves withconsidered as the only available approach for proteome one half being used for biochemical studies and the otheranalysis and several laboratories are constructing 2-DE half for histologic examination. Temporal cortices weredatabases in order to provide them to researchers world used in the present study. The neuropathological assess- 1 wide via the Internet . Although the materials used to ment of AD was made according to the criteria of theconstruct these databases have become more diversified,  Consortium to Establish a Registry for AD  (CERAD) [19].they are still restricted mainly to cultured cells, blood Tissue blocks were dissected and cut into 30  m m wedgecomponents, cardiac muscle, and liver proteins. There is microtome sections. Adjacent sections from the temporalno 2-DE database for human brain proteins presently cortices and the hippocampus of all brains were postfixedavailable on the Internet, which may reflect the hetero- with 10% formaldehyde, and screened to provide a his-geneity of brain tissues, which could produce inconsistent tologic diagnosis. Control brains exhibited negligible2-DE images, and the relatively low reproducibility of microscopic neuropathology (0–2 senile plaques per low2-DE separation of brain proteins. power field). All the AD cases exhibited numerous senileIn the present study, we established a database of brain plaques and neurofibrillary tangles (NFT) throughout theproteins in AD quantified on 2-DE by improving the neocortex.reproducibility of the 2-DE analysis. The most importantstep to permit 2-DE analysis in AD is to establish a  2.2.  Reagents reference map for constructing 2-DE databases. Comparedwith the classical 2-DE using the carrier ampholyte, 2-DE IPG gradient gel strips (pH 4.0–7.0) and Repel-Silaneelectrophoresis using immobilized pH gradients (IPG), were purchased from Pharmacia IPG (Bromma, Sweden).which are an integral part of the polyacrylamide matrix, SDS and 2-D marker were the products of Bio-Rad.has produced significant improvements in 2-DE electro- Phenylmethylsulfonyl fluoride (PMSF) and iodoacetamidephoretic separation, permitting higher resolution and repro- were obtained from Sigma Chemicals (St Louis, MO,ducibility [13–16]. Using IPG for the first dimension of USA). Monoclonal anti- b  actin antibody (clone No. AC-electrophoresis, we applied SDS as a protein solubilization 15) and anti-glial fibrillary acidic protein (GFAP) antibodyreagent for IEF [17].We constructed a reference map using (clone No. G-A-5) were purchased from Sigma Chemicals.Melanie II software (Bio-Rad Inc., Richmond, CA, USA) All other chemicals were obtained from Nakarai (Kyoto,by collecting well-matched spots within selected gels in Japan)order to reduce errors related to the process of making areference gel [18]. With these improvements, we could  2.3.  Sample preparation analyze the protein changes in disease after constructing a2-DE map of human brain proteins. In the present study, Brain tissues were thoroughly sonicated with a handwe sought to establish a 2-DE database by identifying sonicator in 1 v/w of lysis buffer containing 10 mM Trisseveral protein spots on the 2-DE map in control speci- HCl (pH 7.5), 2% SDS, and 2% mercaptoethanol. Aftermens and applied the database to the detection of specific centrifugation at 100 000  g  for 1 h, the supernatant waschanges in these spots in AD. We have made this database collected and diluted with sample buffer containing 9 Mavailable on the Internet for use by all researchers under- urea, 0.5% Triton-X 100, and 0.14% PMSF.taking protein analysis in AD whose collaboration, in turn,should make the 2-DE database complete and useful for  2.4.  The  2  -  DE system exploring the multifaceted disease process involving brainproteins in AD. The first dimension of gel electrophoresis was carriedout using an immobilized pH gradient gel (immobilizeddry strip gel, pH 4–7/18 cm, Pharmacia) with a horizontal 2. Materials and methods  electrophoresis apparatus (Multiphor II, Pharmacia) ac-cording to the method described by Gorg et al. [20]. The 2.1.  Autopsy brain samples  sample solutions were applied on the anodic side of the geland were run according to the manufacturer’s instructions.Brain tissues were obtained at autopsy from 15 patients The second dimension of gel electrophoresis was carrieddiagnosed clinically and histopathologically with AD (63 out on a 15% running gel (20 cm 3 20 cm 3 0.1 cm) in theto 94 years, postmortem period 4 to 21 h), and from 15 presence of SDS essentially as described by Laemmli [21].age-matched controls (60 to 87 years, postmortem period 4 When necessary, marker proteins (SDS-PAGE standardsto 24 h) identified as non-neurological disorder subjects. In and 2D standards from Bio-Rad) were separated in thesame way to estimate the isoelectric points and molecular 1 http://www.expasy.ch; http://biobase.dk/cgi-bin/celis.  weights.  102  T  .  Tsuji et al .  /   Journal of the Neurological Sciences  166 (1999) 100  – 106  2.5.  Protein staining  using a flatbed scanner at 300 dpi (Agfa-Gevaert, Mortsel,Belgium). The image data were analyzed on a MacintoshAfter the second dimension gel electrophoresis, the computer (Power Macintosh 7600/132) using Melanie IIprotein spots were visualized by silver staining using a software (Bio-Rad). We analyzed the spots without fil-Wako silver stain kit II (Wako, Osaka, Japan) which can tering images to avoid artificial effects on images whendetect 15 ng protein on 2-D separated spots. comparisons were made. Spots detected by the programwere matched between each gel in each group, and a 2.6.  Immunoblotting  reference gel was produced by merging the spots from thegels studied. When all of the gels had been matched withImmunodetection is a powerful and sensitive technique the given reference gel, the latter provided a uniquewhich relies on the specificity of antibodies to identify numbering scheme for spot features across all gels. Eachsingle protein spots on 2-D PAGE. Immunoblotting using spot feature in a gel image could then be compared withcommercially available antibodies and the enhanced the corresponding feature in the reference gel. Spotchemiluminescence (ECL) system (Amersham, UK) was features were quantified, including the optical density, areacarried out to identify  b -actin and GFAP on the 2-DE and volume. The volume (VOL) was calculated by thereference map. This enabled the establishment of land- integration of optical density (OD) over the spot’s area,marks to facilitate comparison with other 2-DE reference while the relative volume (%VOL) was the ratio of VOLmaps in future investigations. to total VOL over the whole image. The %VOL of thespots were analyzed to detect specific spots showing 2.7.  Data analysis  significant differences between AD and control groups.Data were analyzed statistically with Statview IV on aProtein spots on silver-stained 2-DE gels were digitized Macintosh, enabling us to identify spots in AD which were Fig. 1. Typical silver-stained image of 2-DE gels in control human brain. The pH range is 4 to 7 and the molecular weight markers represent, from top tobottom, 97.4, 66.2, 45.0, 31.0, 21.5, and 14.5 (kDa). This image was obtained through digitization with ARCUS II (Agfa-Gevaert, Mortsel, Belgium),subsequently processed using the Melanie II program. Without processing of this image, such as subtraction of background staining, the spots are detectedas well-separated areas throughout the gel. By immunoblotting, two groups of spots are identified on the image as  b -actin (single arrow) and GFAP(arrowhead).  T  .  Tsuji et al .  /   Journal of the Neurological Sciences  166 (1999) 100  – 106   103 significantly changed from the controls by one-way analy- were identified. Quantitative analysis using %VOL iden-sis of variance and Bonferroni/Dunnett’s  t  -test, defining tified 28 spots decreased in AD (Fig. 2, Table 1). One of significance as  P , 0.05. these spots was identified as GFAP. 3. Results  3.3.  Protein spots significantly increased in AD brain 3.1.  Detection of protein spots on the gels  Five protein spots were identified which are significantlyincreased in AD brains. The molecular weights of four of A photograph of a 2-D gel, representing the unfiltered these increased spots (213, 215, 221, 226) were approxi-raw image, demonstrated high-resolution separation of mately 52 kDa with isoelectric points ranging from pHspots and low background staining (Fig. 1). About 700 4.44 to 5.04. Two of these four spots were identified asspots were assigned to a synthetic reference gel, including GFAP (Figs. 1 and 2). One low molecular weight proteinwell-matched spots shared with the control group. There (about 3 to 10 kDa) was detected as an increased spot inwere far fewer protein spots heavier than 100 kDa than AD (Fig. 2, Table 1).proteins lighter than 100 kDa. Two individual protein spotswere identified as b -actin (Fig. 1: single arrow) and GFAP(Fig. 1: arrowhead) by immunoblot analysis.  3.4.  Protein spots detected only in AD brain 3.2.  Protein spots lost or significantly decreased in AD  We detected nine spots present in AD brains and absent brain  in controls. These spots were small in volume and faint instaining except for the spots designated A107 and A695No protein spots present in controls but absent in AD (Fig. 2, Table 1). Fig. 2. Profile of spots significantly changed in AD compared with the synthetic reference gel. Gray spots represent proteins unchanged in AD brain. Black spots identified by represent significantly increased ( P , 0.05) proteins in AD brain, and black spots identified by represent significantly decreased( P , 0.05) proteins in AD brain. Black spots identified by H represent proteins detected only on gels in the AD group. Two groups of spots are identifiedin the image as  b -actin (single arrow) and GFAP (arrowhead). Two increased spots and one decreased spot were identified as GFAP.  104  T  .  Tsuji et al .  /   Journal of the Neurological Sciences  166 (1999) 100  – 106  Table 1List of two-dimensional electrophoresis protein spots whose density is changed in the AD temporal cortex compared with control temporal cortex from a patients with non-neurological disordersID p  I   MW OD ratio ID p  I   MW OD ratio(AD/control) (AD/control) PI   /   MW of spots decreased in AD brain PI   /   MW of spots decreased in AD brain 60 6.36 66 0.762 486 4.55 32 0.19774 6.80 65 0.124 505 4.60 31 0.22981 4.55 63 0.216 536 4.29 29 0.84390 4.69 63 0.216 545 5.49 29 0.16095 4.77 62 0.157 572 4.41 29 0.19597 5.26 62 0.199 581 5.57 29 0.672113 5.12 61 0.108 606 4.72 29 0.237131 5.10 59 0.610 716 5.68 12 0.283137 6.41 59 0.138 732 6.42 10 0.236148 5.12 58 0.561 759 5.43 6 0.152201 5.38 53 0.172209 4.55 53 0.465  PI   /   MW of spots increased in AD brain 217 5.14 52 0.809 213 4.68 52 1.294222 4.52 52 0.155 215 4.44 52 1.339260 6.68 49 0.144 221 4.47 52 1.381280 4.54 48 0.701 226 5.04 52 1.455285 5.59 47 0.190 765 5.57 3 1.881297 4.52 47 0.666306 4.52 46 0.656  PI   /   MW of spots detected only in AD brain 307 4.58 46 0.817 A5 6.44 98312 4.65 46 0.795 A10 6.38 98314 6.68 46 0.176 A107 5.18 66317 4.49 45 0.152 A638 4.69 42335 4.49 45 0.752 A695 4.29 38347 4.60 44 0.785 A737 4.44 36357 4.60 43 0.754 A830 4.27 32379 5.24 42 0.152 A877 4.27 31420 5.18 38 0.163 A1023 6.44 28424 5.21 38 0.229 a The list includes the spot identification number (ID), isoelectric point (p  I  ) and molecular weight (MW). Quantification of spots was carried out by%VOL (see Materials and Methods) and the results were analyzed statistically using one-way analysis of variance. Significant differences between AD andcontrol groups was evaluated by Bonfferoni/Dunnett’s  t  -test ( P , 0.05). The figure in the ‘OD ratio (AD/control)’ column is the ratio of the mean value of optical density in AD vs. control. 4. Discussion  focused mainly on the detection of genetic mutation orpost-translational modification of proteins such as tau [27],Analysis with 2-DE has been applied mainly to the APP [28], actin [29] and heat shock proteins [30]. Inproteins of cultured cells, blood components, serum, or studies investigating changes in the expression of brainbody fluids, which are relatively homogeneous [16,18,22]. proteins on 2-DE, Mattila and Frey [31] observed fourSeveral improvements have allowed the application of this protein spots in Alzheimer brains which were differentmethod to human brain, a heterogeneous tissue containing from the controls when they applied IPG to the firstmany cellular components such as a variety of neurons and dimension of electrophoresis; one spot was undetectable,glia as well as microvessels. Improvement in obtaining two spots were significantly weaker and one spot waswell-resolved and highly reproducible gel images was stronger than those in controls. Our present results in-achieved using IPG for the first dimension of electro- cluded several more spots which were significantlyphoresis [23,24]. In the present study, reference gels were changed in AD, probably because we used a narrow pHcreated by automatically merging a set of gel images that range (pH 4 to 7) and a large (180 3 180 mm) secondcontained at least three pairwise-matched gels. In spite of dimension SDS gel, which could result in better separation.factors such as autopsy delay, conditions of sonication, and However, we encountered several unresolved problems inthawing in the equilibration solution that might influence the separation of proteins. We were unable to demonstrateresults, the analysis system provided the possibility of proteins of molecular weight greatly exceeding 100 kDa,extracting data from a reliable standard spot in often which might be due to the limited ability of heavy proteinsvariable 2-DE images; this was achieved using Melanie II to enter the first dimension IPG gel. Studies includingas the data analysis program [25,26]. larger numbers of cases and the investigation of moreApplication of 2-DE analysis in AD research has been acidic and basic proteins will be necessary. However, the
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