changeshippocampalproteinpostnatalbraindevelopmentrat_06

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Changes of Hippocampal Protein Levels during Postnatal BrainDevelopment in the Rat

Rachel Weitzdo 1 rfer, Harald Ho 1 ger, Arnold Pollak, and Gert Lubec* ,

Medical University of Vienna, Department of Neonatology, Vienna, Austria, and Core Unit of Biomedical Research, Division of Laboratory Animal Science and Genetics, Medical University of Vienna, Austria

Received May 26, 2006

Abstract: Information on postnatal brain protein expressionis very limited, and we therefore compared hippocampalprotein levels in rat hippocampus at different develop-mental time points using two-dimensional gel electro-phoresis followed by mass spectrometrical protein iden-tification and specific software for quantification. Proteinsfrom several cascades as e.g., antioxidant, metabolic,

cytoskeleton, proteasomal, and chaperone pathways weredevelopmentally regulated, which is relevant for designand interpretation of protein chemical studies in themammalian brain.

Keywords: temporal regulation rat hippocampus postnataldevelopment MALDI brain development age-dependentprotein expression

Introduction

Gene expression in the brain is still holding center stage andspringing surprises. The advent of proteomics technology,

however, now challenges systematic studies at the proteinlevel. 1 Although there is abundant information on develop-mentally regulated individual proteins in human and rodentbrain, a systematic study on developmental brain protein levelsduring the postnatal period has not been carried out so far.Fountoulakis and co-workers described differences in proteinlevels between neonatal and adult rat brain 2 using two-dimensional gel electrophoresis with mass spectrometricalidentification of proteins revealing a series of temporally expressed proteins. Tsugita et al. reported spatial and temporalexpression of mouse brain proteins from the 10th week untilthe 24th month of age and carried out protein profiling in ratcerebella during development 3 using comparable technology. 4

Several proteins from different protein pathways were reported

to show temporally regulated brain protein levels. The effectof aging on mouse pituitary protein levels were revealed by Marzban et al. 5 by two-dimensional gel electrophoresis andN-terminal micro-sequencing. Fluorescent difference two-dimensional gel electrophoresis followed by mass spectro-metrical analysis of kitten and cat visual cortex showed

differential protein levels between adult and 30 day old kittens. 6

In addition, protein profiling was carried out in a series of different brain areas from several species, without comparisonto other stages. 7- 9

However important these reports are, no high-throughputtechnology was applied and results from the studies abovecannot be extrapolated to the rat. A systematic study of brainprotein levels during the postnatal period seems mandatory as protein hallmarks of brain development would be of importance for understanding neurobiology and neuropathol-ogy.

We therefore aimed to study brain protein levels at threedevelopmental time points in a widely used and well-characterized rat strain with representative sample sizes. Forthis purpose a nonsophisticated proteomic approach, two-dimensional gel electrophoresis with subsequent mass spec-trometrical identification of proteins and quantification withspecific software was used. Herein we report protein profiling and differential brain protein levels of several protein classesin the rat thus extending and confirming knowledge ondevelopmental regulation of brain proteins and indeed, mostof these proteins were not shown to be temporally regulatedso far. We have been selecting the hippocampus as this is akey area for cognitive functions and can be well-dissected inthe rat and protein expression in this area is of pivotal interestto a broad neuroscientific forum.

Experimental Section Animals. Three day, three week, and three month old female

Sprague - Dawley rats (Institute of Animal Breeding, University of Vienna, Himberg, Austria) were housed in groups of up tosix per cage. Rats were maintained on 11/13 h light/dark cyclein a temperature (21 ( 1 C) and humidity (50 ( 10%)controlled and well-ventilated room with access to food anddrink ad libitum. The animals were bred and kept underspecific pathogen free (SPF) conditions, and all of the experi-ments were carried out in accordance with the rules of the American Physiology Society. Animals were sacrificed by decapitation, brains were rapidly removed and complete hip-pocampal tissue was taken within one minute, snap frozen,and stored at - 80 C until chemical analysis, and the freezing chain was never interrupted. 10

The rational to select these three age groups was that theseare widely used for biochemical, genetic, and pharmacologicalstudies.

Sample Preparation. Hippocampal tissue samples ( n ) 10hippocampi per group, pooled left and right hippocampus from

* To whom correspondence should be addressed. Prof. Dr. Gert Lubec,Medical University of Vienna, Department of Pediatrics, Wahringer Gurtel18, A-1090 Vienna, Austria; Tel: + 43-1-40400-3215 Fax: + 43-1-40400-3194;E-mail: [email protected].

Medical University of Vienna. Core Unit of Biomedical Research.

10.1021/pr0602545 CCC: $33.50 2006 American Chemical Society Journal of Proteome Research 2006, 5, 3205 - 3212 3205Published on Web 10/06/2006


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10 rats each that were not littermates) were homogenized andsuspended in 1.8 mL of sample buffer consisting of 8 M urea(Merck, Darmstadt, Germany), 2 M thiourea (Sigma, St. Louis,MO), 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate) (Sigma, St. Louis, MO), 65 mM 1,4-dithioerythritol (Merck, Germany), 1mM EDTA (ethylenedia-mintetraacetic acid), 1 mM PMSF, and 0.5% carrier ampholytesResolyte 3.5 - 10 (BDH Laboratory Supplies, Electran, England).Samples were left at room temperature for 1 h and then

centrifuged at 14 000

g for 60 min, and the supernatant wastransferred into Ultrafree-4 centrifugal filter units (Millipore,Bedford, MA) for desalting and concentrating proteins. 11,12

Protein content of the supernatant was quantified by theBradford protein assay system. 13

Two-Dimensional Gel Electrophoresis (2-DE). 2 DE wasperformed as reported: 14 Samples of 800 g protein were appliedon immobilized pH 3 - 10 nonlinear gradient strips in samplecups at their basic and acidic ends. Focusing was started at200 V, and the voltage was gradually increased to 8000 V at 4 V/min and then kept constant for a further 3 h (approximately 150 000 Vh totally). After the first dimension, strips (18 cm) wereequilibrated for 15 min in the buffer containing 6 M urea, 20%glycerol, 2% SDS, 2% DTT, and then for 15 min in the same

buffer containing 2.5% iodoacetamide instead of DDT. Afterequilibration, strips were loaded on 9 - 16% gradient sodiumdodecyl sulfate polyacrylamide gels for second-dimensionalseparation. The gels (180 200 1.5 mm 3) were run at 40 mA per gel. Immediately after the second dimension run, gels werefixed for 12 h in 50% methanol, containing 10% acetic acid,and the gels were stained with Colloidal Coomassie Blue(Novex, San Diego, CA) for 12 h on a rocking shaker. Molecularmasses were determined by running standard protein markers(Biorad Laboratories, Hercules, CA) covering the range 10 - 250kDa. p I values were used as given by the supplier of theimmobilized pH gradient strips (Amersham Bioscience, Upp-sala, Sweden). Excess of dye was washed out from the gels withdistilled water, and the gels were scanned with ImageScanner

(Amersham Bioscience).Electronic images of the gels were recorded using Adobe

Photoshop and Microsoft Power Point Softwares.Quantification of Protein Spots. Protein spots were outlined

(first automatically and then manually) and quantified using the ImageMaster 2D Elite software (Amersham Biosciences,Uppsala, Sweden). The percentage of the volume of the spotsrepresenting a certain protein was determined in comparison with the total proteins present in the 2-DE gel. 15

Matrix-Assisted Laser Desorption Ionization Mass Spec-trometry. Spots were excised with a spot picker (PROTEINEERsp, Bruker Daltonics, Germany), placed into 384-well microtiterplates and in-gel digestion and sample preparation for MALDIanalysis were performed by an automated procedure (PRO-TEINEER dp, Bruker Daltonics). 16 Briefly, spots were excisedand washed with 10 mM ammonium bicarbonate and 50%acetonitrile in 10 mM ammonium bicarbonate. After washing,gel plugs were shrunk by the addition of acetonitrile and driedby blowing out the liquid through the pierced well bottom. Thedried gel pieces were reswollen with 40 ng/L of trypsin(Promega, U.S.A.) in enzyme buffer (consisting of 5 mM octyl-D-glucopyranoside (OGP) and 10 mM ammonium bicarbonate)and incubated for 4 h at 30 C. Peptide extraction wasperformed with 10 L of 1% TFA in 5 mM OGP. Extractedpeptides were directly applied onto a target (AnchorChip,Bruker Daltonics) that was loaded with R -cyano-4-hydroxy-

cinnamic acid (Bruker Daltonics) matrix thinlayer. The massspectrometer used in this work was an Ultraflex TOF/TOF(Bruker Daltonics) operated in the reflector mode for MALDI-TOF peptide mass fingerprint (PMF) or LIFT mode for MALDI-TOF/TOF fully automated using the FlexControl software. Anaccelerating voltage of 25 kV was used for PMF. Calibration of the instrument was performed externally with [M + H] + ionsof angiotensin I, angiotensin II, substance P, bombesin, andadrenocorticotropic hormones (clip 1 - 17 and clip 18 - 39). Each

spectrum was produced by accumulating data from 200consecutive laser shots. Those samples that were analyzed by PMF from MALDI-TOF and were significantly different betweengroups were additionally analyzed using LIFT-TOF/TOF MS/MS from the same target. A maximum of three precursor ionsper sample were chosen for MS/MS analysis. In the TOF1 stage,all ions were accelerated to 8 kV under conditions promoting metastable fragmentation. After selection of jointly migrating parent and fragment ions in a timed ion gate, ions were liftedby 19 kV to high potential energy in the LIFT cell. After furtheracceleration of the fragment ions in the second ion source, theirmasses could be simultaneously analyzed in the reflector withhigh sensitivity. PMF and LIFT spectra were interpreted withthe Mascot software (Matrix Science Ltd, London, UK). Data-

base searches, through Mascot, using combined PMF and MS/MS datasets were performed via BioTools 2.2 software (Bruker). A mass tolerance of 25 ppm and 1 missing cleavage site forPMF and MS/MS tolerance of 0.5 Da but no missing cleavagesite for MS/MS search were allowed and oxidation of methion-ine residues was considered. The probability score calculatedby the software was used as criterion for correct identification.

The algorithm used for determining the probability of a falsepositive match with a given mass spectrum is describedelsewhere. 17

Results

A series of 190 proteins were identified in the three groupsby MALDI-TOF, and identification of statistically significantdifferentially expressed proteins was verified by MALDI-TOF/TOF.

The significantly differentially expressed proteins were fromseveral protein classes and pathways. Antioxidant, metabolic,cytoskeleton, proteasome, nucleic acid binding proteins as wellas proteins from chaperones were temporally regulated.

The statistical evaluation of differences between groups wascarried out either by Fishers exact test (Table 1a), as severalproteins were undetectably low in the individual groups, or by ANOVA followed by appropriate post-hoc tests (Table 1b).

As shown in Table 1a, proteins from metabolism, cytoskel-eton, and the protein synthetic and handling machinery weredifferentially expressed. Results are expressed as number of present spots for an individual protein per group.

In Table 1b, means and standard deviation are givenrevealing that proteins from several classes as shown above,including antioxidant proteins, were temporally expressed.

In Table 2, information on identification of significantly expressed proteins is listed. Results from nonsignificantly different protein levels are shown in Table 3a,b (Supporting Information). Information on the identification of nonsignifi-cantly and differentially expressed proteins is provided insupplementary Table 4 (Supporting Information). The corre-sponding images/maps are presented in Figure 1. The stringentconditions for considering the level of significance as P < 0.001 were selected for correcting false positives by multiple testing.

Postnatal Brain Protein Expression technical notes

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Table 1.

a. Temporally Significant Expression of Proteins (Fishers Exact Test)

accessionnumber protein name 3 d 3 w 3 m 3d vs 3w 3d vs 3m 3w vs 3m

Metabolic proteinsP25113 - 1 phosphoglycerate mutase 1 (rat) 0 a/9 10/10 9/9 < 0.0001 < 0.0001 nsP27139 carbonic anhydrase II (rat) 0/9 0/10 8/8 ns < 0.0001 < 0.0001P50516 vacuolar ATP synthase catalytic subunit A,

ubiquitous isoform (mouse)0/9 3/10 9/9 0.0007 < 0.0001 ns

P80254 D-dopachrome tautomerase (rat) 0/9 10/10 8/8 < 0.0001 < 0.0001 0.0044

Q99NA5 NAD+

-specific isocitratedehydrogenase a-subunit 0/9 7/7 8/8 0.0001 0.0001 nsCytoskeleton proteins

P39053 - 1 dynamin-1 (mouse) 0/9 10/10 8/8 < 0.0001 < 0.0001 nsP39053 - 2 dynamin-1 (mouse) 0/8 10/10 9/9 < 0.0001 < 0.0001 nsP39053(total of 2)

dynamin-1 (mouse) 0/9 10/10 9/9 < 0.0001 < 0.0001 ns

ProteasomeO35593 -total spot volume

26S proteasome,non-ATPase subunit

9/9 9/9 0/8 < 0.05 < 0.0001 < 0.0001

P60901 proteasome subunit alpha type 6 (human) 0/9 9/9 7/8 < 0.0001 < 0.0001 nsP99026 proteasome subunit beta

type 4 [Precursor] (mouse)0/9 8/8 7/7 < 0.0001 < 0.0001 ns

Nucleic acid binding proteinsO35737 - 3 heterogeneous nuclear

ribonucleoprotein H (mouse)0/9 0/8 7/7 ns < 0.0001 < 0.0001

O35737 - 4 heterogeneous nuclearribonucleoprotein H (mouse)

0/9 0/9 9/9 ns < 0.0001 < 0.0001

P70333 heterogeneous nuclear

ribonucleo-protein H (mouse)

0/9 9/9 9/9 < 0.0001 < 0.0001 ns

Q9D6G1 85% identical to heterogeneous nuclearribonucleo-protein A/B

0/9 9/9 8/8 < 0.0001 < 0.0001 ns

ChaperonesP11598 protein disulfide isomerase

A3 [Precursor] (rat)0a/9 7/7 0/7 < 0.0001 ns < 0.0001

b. Temporally Significant Expression of Proteins

3 d 3 w 3 monthsaccessionnumber protein name N mean SD N mean SD N mean SD ANOVA 3d vs 3w 3d vs 3m 3w vs 3m

Antioxidant proteinsO35244 peroxiredoxin 6 (rat) 9 0.117 0.037 10 0.397 0.076 9 0.279 0.081 < 0.0001 < 0.001 c < 0.05 c ns c P04905 glutathione S-transferase Yb-1 9 0.040 0.042 0 e - - 8 0.137 0.085 - 0.0002 d -P04906 glutathione S-transferase P (rat) 9 0.181 0.050 9 0.036 0.028 9 0.115 0.054 < 0.0002 < 0.001 c ns c ns c P07632 superoxide dismutase [Cu - Zn]

(rat)8 0.263 0.043 10 0 .546 0 .184 7 0.850 0 .083 0 .0001 ns c < 0.001 c ns c

Metabolic proteinsP11980 - 1 pyruvate kinase, M1 isozyme 9 0.087 0.024 9 0.144 0.08 8 0.37 0.12 0.0003 ns c < 0.001 c < 0.05 c P11980 - 2b pyruvate kinase, M1 isozyme 9 0.29 0.084 9 0.351 0 .096 8 0.958 0 .493 0.0013 ns c < 0.01 c < 0.05 c P11980 - 3b pyruvate kinase, M1 isozyme 8 0.076 0.019 9 0.036 0 .046 8 0.161 0 .125 0.0206 ns c ns c < 0.05 c P11980(total of 3)

pyruvate kinase, M1 isozyme 9 0.444 0.104 9 0.531 0 .187 8 1.488 0 .661 0.0004 ns c < 0.001 c < 0.01 c

P23492 purine nucleoside phosphorylase 9 0.341 0.083 9 0.234 0.101 8 0.129 0.043 0.0007 ns c < 0.001 c ns c P25113 - 2 phosphoglycerate mutase 1 (rat) 9 0.245 0.069 9 0.609 0.114 8 0.722 0.108 0.0001 < 0.01 c < 0.001 c ns c P25113(total of 2) b

phosphoglycerate mutase 1 (rat) 9 0.245 0.069 9 0.733 0.14 8 0.915 0.098 < 0.0001 < 0.05 c ns c ns c

P31399 ATP synthase D chain,mitochondrial (rat)

8 0.235 0.073 9 0.724 0 .117 9 0.825 0 .239 0.0002 < 0.01 c < 0.001 c ns c

P48500 - 1 triose-phosphate isomerase (rat) 9 0.303 0.043 9 0.122 0.054 9 0.217 0.066 0.0002 < 0.001 c ns c ns c P48500 - 2 triose-phosphate isomerase (rat) 9 0.137 0.035 10 0.295 0.121 9 0.763 0.26 0.0001 ns c < 0.001 c < 0.05 c P48500 - 3b triose-phosphate isomerase (rat) 9 0.239 0.119 10 0.273 0.082 9 0.367 0.196 ns ns c ns c ns c P48500(total of 3) b

triose-phosphate isomerase (rat) 9 0.679 0.157 10 0.678 0.201 9 1.346 0.478 0.0012 ns c < 0.01 c < 0.01 c

P97532 3-mercapto-pyruvatesulfur-transferase (rat)

9 0.028 0.055 10 0 .135 0 .052 9 0.082 0 .043 0 .0007 0.0007 c 0.0023 c ns c

Cytoskeleton proteinsO89053 coronin 1A 8 0.259 0.096 10 0.558 0.115 8 0.541 0.052 0.0005 < 0.001 c < 0.01 c ns c P11516 lamins C and C2 (mouse) 8 0.373 0.144 10 0.076 0.038 0 e - - 0.0003 d - -Q61553 - 1 fascin (mouse) 9 0.239 0.099 10 0.031 0.053 8 0.069 0.042 0.0003 < 0.001 c < 0.05 c ns c Q61553 - 2 fascin (mouse) 9 0.522 0.136 9 0.334 0.084 7 0.195 0.058 0.0002 ns c < 0.001 c ns c Q61553 - 3b fascin (mouse) 9 0.177 0.072 9 0.085 0.024 8 0.126 0.027 0.0048 < 0.01 c ns c ns c Q61553(total of 3)

fascin (mouse) 9 0.938 0.184 9 0.455 0.095 7 0.395 0.053 0.0002 < 0.01 c < 0.001 c ns c

ProteasomeQ00981 -total spotvolume

ubiquitin carboxyl-terminalhydrolase isozyme L1 (rat)

8 1.877 0.165 9 0.774 0 .408 9 1.458 0 .498 0.0006 < 0.001 c ns c ns c

Q9JHW0 proteasome subunit betatype 7 [Precursor] (rat)

9 0.293 0.063 8 0.174 0.036 9 0.126 0.035 0.0002 ns c < 0.001 c ns c

Q9R1P4 proteasome subunit alphatype 1

9 0.33 0.086 8 0.214 0.112 8 0.129 0.043 0.0016 ns c < 0.001 c ns c

technical notes Weitzdo 1 rfer et al.

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Discussion

As shown in the Results section, several proteins of individualprotein pathways were shown to be developmentally regulated.Statistical analysis revealed significantly differential expressionof proteins and some structures were not even detectable in

the hippocampus of early postnatal life, as shown by Fishersexact test. The major finding of this study is the demonstrationof proteins that have not been previously shown to bedevelopmentally regulated, neither in the rodent nor in thehuman system. Temporal regulation of a few proteins was

Table 1. (Continued)

3 d 3 w 3 monthsaccessionnumber protein name N mean SD N mean SD N mean SD ANOVA 3d vs 3w 3d vs 3m 3w vs 3m

Nucleic acid binding proteinsO35737 - 1b heterogeneous nuclear

ribonucleoprotein H (mouse)7 0.122 0.043 8 0.042 0.013 8 0.06 0.02 0.0017 < 0.01 c ns c ns c

O3573thr7 - 2 heterogeneous nuclearribonucleoprotein H (mouse)

9 1.033 0 .231 9 0.397 0.167 9 0.364 0.073 0.0002 < 0.01 c < 0.001 c ns c

O35737(total of 4)

heterogeneous nuclearribonucleoprotein H (mouse)

9 1.128 0 .242 9 0.435 0.172 9 0.553 0.086 0.0001 < 0.001 c < 0.01 c ns c

O88569 - 1 heterogeneous nuclearribonucleoproteins A2/B1 8 0.508 0.091 7 0.244 0.05 8 0.092 0.042 < 0.0001 ns c < 0.001 c ns c

O88569 - 2 heterogeneous nuclearribonucleoproteins A2/B1

8 0.512 0.149 7 0.161 0.03 6 0.051 0.019 0.0001 ns c < 0.001 c ns c

O88569(total of 2)

heterogeneous nuclearribonucleoproteins A2/B1

8 1.02 0.151 7 0.405 0.06 8 0.13 0.024 < 0.0001 ns c < 0.001 c ns c

P42669 transcriptional activatorprotein PUR-alpha (mouse)

8 0.131 0.022 9 0.32 0.067 9 0.264 0.097 0.0003 < 0.001 c < 0.05 c ns c

Q9CT01 heterogeneous nuclearribonucleoprotein D-like

8 0.377 0 .045 8 0.163 0.051 8 0.148 0.039 0.0004 < 0.01 c < 0.001 c ns c

ChaperonesP28480 - 1b T-complex protein 1,

alpha subunit (rat)8 0.095 0.044 8 0.06 0.02 8 0.044 0.025 0.0454 ns c < 0.05 c ns c

P28480 - 2 T-complex protein 1,alpha subunit (rat)

8 0.872 0.31 8 0.45 0.086 8 0.174 0.058 < 0.0001 ns c < 0.001 c < 0.05 c

P28480(total of 2)

T-complex protein 1,alpha subunit (rat)

8 0.967 0.338 8 0.51 0.092 8 0.218 0.071 < 0.0001 ns c < 0.001 c < 0.05 c

a Zero means undetectably low. b Belongs to a group of significant proteins. c Post-hoc test. d Mann Whitney-U test. e Technically not quantifiable.

Table 2. Identification of Significant Proteins

number protein name matches score p I (kDa)

Antioxidant proteinsO35244 peroxiredoxin 6 (rat) 25 198 5.65 24.68P04905 glutathione S-transferase Yb-1 13 96 8.42 25.75P04906 glutathione S-transferase P (rat) 10 149 6.89 23.43P07632 superoxide dismutase [Cu - Zn] (rat) 7 61 5.89 15.78

Metabolic proteinsP11980 pyruvate kinase, M1 isozyme (rat) 32 474 6.69 57.68P23492 purine nucleoside phosphorylase 16 85 5.78 32.277P25133 phosphoglycerate mutase 1 (rat) 18 112 6.21 28.51P27139 carbonic anhydrase II (rat) 11 182 6.88 28.98P31399 ATP synthase D chain, mitochondrial (rat) 13 123 6.21 18.63P48500 triosephosphate isomerase (rat) 19 255 6.51 26.78P50516 vacuolar ATP synthase catalytic subunit A, ubiquitous isoform (mouse) 31 189 5.62 68.26

P80254 D-dopachrome tautomerase (rat) 9 81 6.15 13P97532 3-mercaptopyruvate sulfurtransferase (rat) 11 68 5.88 32.8Q00NA5 NAD+ -specific isocitrate dehydrogenase a-subunit 37 190 6.46 39.61

Cytoskeleton proteinsO89053 coronin 1A 24 142 6.05 50.98P11516 lamins C and C2 (mouse) 22 112 6.05 65.4P39053 dynamin-1 (mouse) 32 228 7.61 97.8Q61553 fascin (mouse) 35 269 6.21 54.4

ProteasomeO35593 26S proteasome, non-ATPase subunit 14 19 73 6.18 34.56P60901 proteasome subunit alpha type 6 (human) 15 117 6.35 27.39P99026 proteasome subunit beta type 4 [precursor] (mouse) 12 70 5.47 29.11Q00981 ubiquitin carboxyl-terminal hydrolase isozyme L1 (rat) 12 84 5.12 24.77Q9JHW0 proteasome subunit beta type 7 [precursor] (rat) 8 65 8.14 29.92Q9R1P4 proteasome subunit alpha type 1 (mouse) 11 177 6 29.54

Nucleic acid binding proteinsO35737 heterogeneous nuclear ribonucleoprotein H (mouse) 11 381 5.89 49.19O88569 heterogeneous nuclear ribonucleoproteins A2/B1 (mouse) 28 252 8.67 35.99P42669 transcriptional activator protein PUR-alpha (mouse) 24 86 6.07 34.88P70333 heterogeneous nuclear ribonucleoprotein H (mouse) 19 72 5.89 49.27Q9CT01 heterogeneous nuclear ribonucleoprotein D-like 11 114 4.9 16.53Q9D6G1 85% homologous to heterogeneous nuclear ribonucleoprotein A/B (acc. no: Q99020) 15 119 6.07 29.92

ChaperonesP11598 protein disulfide isomerase A3 [precursor] (rat) 27 263 5.88 56.62P28480 T-complex protein 1, alpha subunit (rat) 39 279 5.86 60.35

Postnatal Brain Protein Expression technical notes

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Figure 1. (a - f) Master gels representing maps of antioxidant, metabolic, cytoskeleton, proteasome, nucleic acid binding, and chaperoneproteins in three months old rat hippocampus, stained with Coomassie blue are presented and developmentally regulated proteinsare highlighted in black boxes. Brains of 3 day (d), 3 week (w), and 3 month (m) old rats were used in the study.

technical notes Weitzdo 1 rfer et al.

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already shown before, and these observations, obtained at thetranscriptional level or by immunochemical techniques, arenow confirmed by a proteomic approach.

Among several antioxidant proteins that showed comparablehippocampal protein levels, glutathion-S-transferase P wasabundant in the 3 day old (3d) rat pups in contrast toglutathione-S-transferase Yb-3. Unfortunately, results at themRNA steady-state level/transcripts in the rat 18 cannot beextrapolated to the findings at the protein level due to several

technical reasons, and the whole brain rather than just thehippocampus was used in the previous study. This problem isinherent as most authors have been reporting systematic anddevelopmental regulation of proteins using the whole brain.Moreover, the majority of studies has been focusing on fetalbrain development rather than postnatal brain development, which, indeed, formed one rational to put the emphasis onpostnatal stages. Likewise, no systematic study on postnatalSOD1 expression in brain has been performed, and data areavailable in other organs at the enzyme activity level only. 19

Herein we reveal an increase of SOD1 in adult rat hippocampusalthough the biological meaning remains elusive; these findingsare relevant when future studies at the protein level within theperiod studied are designed. It may be speculated that regula-

tion of antioxidant proteins are linked to well-known anddocumented metabolic changes in the brain during this periodof early life. Two more, peroxiredoxin 6 and glutathioneS-transferase Yb-1, were under developmental control.

Among a long list of metabolic proteins, only a couple of proteins from energy, intermediary, carbohydrate, transport,and amino acid metabolism were under age-dependent control.

Key elements of carbohydrate metabolism revealed differentlevels, probably reflecting metabolic rates at the corresponding time points. It is, however, evenly important that so-calledhouse-keeping genes (proteins), used for normalization of results, vary with postnatal age and may therefore represent aconfounding factor in protein expression studies. 20

Likewise, pyruvate kinase M1 isoenzyme may reflect age-dependent intermediary and purine nucleoside phosphorylaseage-dependent purine metabolism.

3-Mercaptopyruvate sulfurtransferase, on the other hand,turned out to be lowest in 3d old rat pups, probably indicating that cysteine metabolism/degradation i.e., the conversion fromcysteine in the rat pup by transsulfuration from mercaptopy-ruvate to pyruvate, may not be a major trait in early postnatallife.21 This differential protein level in the hippocampus may,however, not reflect genetically determined protein expressionbut could be due to nutritional factors, as in this age groupdiet is clearly distinct from 3 weeks or 3 months. This statementmay be relevant for other protein levels observed in our andother studies.

As to intermediary metabolism, NAD + specific isocitratedehydrogenase was not detectable in 3d hippocampus, and thismay be interpreted as given above, and no developmental dataare available from literature searches to the best of ourknowledge.

Carbonic anhydrase II (CAII) was not detectable in 3d and3w old rats, and the interpretation of this finding may differfrom above: CAII is the major isoenzyme in the brain partici-pating in acid - base homeostasis, fluid transport, and myelinsynthesis. 22 Although altered fluid transport and imbalances of acid - base homeostasis within the first days of life may beassociated with low CAII levels in 3d rats, a more plausibleexplanation for 3w and 3m is the genetically determined delay

in myelination in the rodent, 23 and CAII levels are known tobe associated and strongly linked to myelination.

Mitochondrial ATP synthase, a representative component of energy metabolism, was only significantly increasing at threemonths although a trend to increase ( P < 0.01) was observed.The stringent statistical difference in the present study defining the level of significance at P < 0.001 is the reason for simply defining a trend of ATP synthase at P < 0.01 in the 3w animals. The different energy requirements in different age

groups of our panels studied, nutritional equivalents, and basicmetabolic rate may be determining factors for low levels at 3d.Bates and co-workers 24 described that in postnatal developmentcomplexes of the electron transport chain in isolated rat brainmitochondria was increasing with age and this finding is inagreement with our observation. This is also in agreement withthe finding of age-controlled vacuolar ATP synthase, acting ina different compartment.

D-Dopachrome tautomerase, cloned by Kuriyama et al., 25 isinvolved in catecholamine metabolism 26. The gradual increasein monoaminergic neurotransmitters 27 may be linked to D-dopachrome tautomerase and may consequently help tointerpret the undetectably low levels at 3d.

Out of the many cytoskeleton proteins, only two fascin

expression forms were developmentally regulated. Three fascinexpression forms were observed and we did not discriminate whether they represented post-translational modifications orsplice variants.

Two fascin spots were significantly more abundant at P

changeshippocampalproteinpostnatalbraindevelopmentrat_06

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