Strain-dependent expression of signaling proteins in the mouse hippocampus

Neuroscience 138 (2006) 149 –158

STRAIN-DEPENDENT EXPRESSION OF SIGNALING PROTEINS IN THE MOUSE HIPPOCAMPUS D. D. POLLAK,a J. JOHN,a A. SCHNEIDER,a H. HOEGERb AND GERT LUBECa*

Individual inbred mouse strains differ from each other in behavior, learning and memory (Crawley et al., 1997; Upchurch and Wehner, 1989), long-term potentiation (LTP), a type of synaptic plasticity that may underlie some forms of learning and memory (Nguyen et al., 2000; Gerlai, 2002) and hippocampal gene expression (Fernandes et al., 2004). Therefore, results obtained from cognitive and behavioral studies using one specific strain cannot be simply extrapolated or compared. The actual situation is even more complex: the use of genetically engineered mice for such studies may be hampered by their genetic background, harboring genomes from several mouse strains and problems with interpretation of results may arise if the background is not corrected by appropriate controls. In addition, knowledge on the “protein chemical background” tentatively underlying synaptic plasticity and neuronal information storage (NIS), in the individual strains is limited (e.g. Fordyce et al., 1994; Bowers et al., 1995). The importance for addressing strain distribution at the proteome level is further underscored by the difficulty in predicting protein characteristics from genomic sequence data alone. These characteristics include posttranslational modifications, subcellular distribution, stability, biomolecular interactions and function. Notably, several diverse mechanisms can result in many protein variants from the same locus in one species: single nucleotide polymorphisms, gene splicing, alternative splicing of pre-mRNA, RNA editing, translational frame shifts and posttranslational modifications. It has become clear that LTP and long-term depression (LTD) have many forms which can be distinguished on the basis of their underlying signaling mechanisms (Grant and O’Dell, 2001). Synaptic plasticity induced following N-methyl-D-aspartate (NMDA)-type glutamate receptor activation and signaling cascades involved therein is well-studied. Recognition that NMDAR channels are highly permeable to Ca2⫹ led to discovery that Ca2⫹ dependent signaling molecules, such as protein kinase C and Ca2⫹/calmodulin kinase II (CaMKII) may play an essential role for synaptic plasticity and NIS. The findings that tyrosine kinase activity is also required for LTP induction (Grant and O’Dell, 2001) and that mitogen-activated protein kinase (MAPK) is activated by NMDAR raise new questions over the complexity of signaling pathways involved in NIS. Since more than 100 molecules have been implicated in LTP-induced synaptic plasticity (Sanes and Lichtman, 1999) it is challenging to understand the relationships and composition of these signaling networks (Grant and O’Dell, 2001). Recently proteomics has been proven a valuable tool for the analysis of signaling proteins specifically with regard to their proposed

a Department of Pediatrics, Division of Pediatric Neuroscience, Medical University of Vienna, Währinger Gürtel 18, A-1090 Vienna, Austria b Core unit of Biomedical Research, Division of Laboratory Animal Science and Genetics, Medical University of Vienna, Brauhausgasse 34, A-2325 Himberg, Austria

Abstract—Individual mouse strains may differ significantly in terms of behavior and cognitive function. Hippocampal gene expression profiling on several mouse strains has been carried out and points toward substantial strain-specific variation of more than 200 genes including components of major signaling pathways involved in neuronal information storage. Strain-specific hippocampal protein expression, however, has not been investigated yet. A proteomic approach based on two-dimensional gel electrophoresis coupled with mass spectrometry has been chosen to address this question by determining strain-dependent expression of signaling proteins in hippocampi of four inbred and one outbred mouse strain. Forty-six spots corresponding to 37 different signaling proteins have been analyzed and quantified. Statistical analysis revealed strain-dependent expression of serine/threonine protein phosphatase 1, serine/threonine protein phosphatase 2A, large GTP binding protein OPA1, guanine nucleotide-binding protein beta, putative GTP-binding protein Ran, receptor of activated protein kinase C1, WASP-family protein member 1, voltage-dependent anion channel 2 and 14-3-3 protein gamma. Differential expression of signaling proteins in the hippocampus may contribute to the molecular understanding of strain-dependent behavioral and cognitive performance. Moreover, these data highlight the importance of the genetic background for the analysis of signaling pathways in the hippocampus in wild-type mice as well as in gene-targeting experiments. © 2005 Published by Elsevier Ltd on behalf of IBRO. Key words: protein expression, brain, inbred mouse strain, proteomics, synaptic plasticity. *Corresponding author. Tel: ⫹43-1-40400-3215; fax: ⫹43-1-404003194. E-mail address: [email protected] (G. Lubec). Abbreviations: Balb, Balb/c; C57, C57BL/6J; CaMKII, Ca2⫹/calmodulin kinase II; DTT, 1,4-dithioerythritol; EDTA, ethylenediaminetraacetic acid; FVB, FVB/N; GNB1, guanine nucleotide-binding protein beta subunit 1; LTP, long-term potentiation; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; MAPK, mitogen-activated protein kinase; NIS, neuronal information storage; NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptor; OF, OF1; OGP, ␤-D-glucopyranoside; PMF, peptide mass fingerprint; PP, protein phosphatase; PP1, protein phosphatase 1; PP1-beta, serine/threonine protein phosphatase 1, beta catalytic subunit; PP2A, protein phosphatase 2A; RACK1, receptor of activated protein kinase C 1; VDAC, voltage-dependent anion channel; WAVE-1 protein, WASP-family protein member 1; 129Sv, 129S2/Sv; 2-DE, two-dimensional gel electrophoresis. 0306-4522/06$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.11.004

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role in mediating activity-dependent synaptic plasticity (Yamauchi, 2002; Collins et al., 2005). Although a vast variety of signaling proteins and their relevance for NIS have been studied so far, strain-specific expression of these structures has not yet been systematically evaluated and we therefore decided to carry out the present study. The aim of this study was to complement the currently available gene expression data at the protein level by focusing on proteins representing candidate molecules involved in neuronal processes underlying basic brain functions. A systematic comparison of wild-type strains should provide some insight into the regulation of basic physiological brain processes as well as the mechanisms potentially contributing to strain-specific phenotypes. Four inbred strains (FVB/N, C57Bl/6J, 129S2/Sv and Balb/c), commonly used for generating genetically modified mice and for conventional experiments in pharmacology and toxicology and one outbred strain (OF1) were selected for studying the expression of signaling proteins in the hippocampus, a region know to be essential for NIS and storage. We have taken this approach further by carefully constructing a data set of hippocampal protein expression profiles specific for a defined brain function, providing a comprehensive tool for candidate protein identification in behavior. Genetically driven expression differences detected from the study can then be related to phenotypic differences among these inbred strains of mice, thus nominating candidate genes that are functional in the sense that they yield expression differences between the strains (Fernandes et al., 2004), although much larger panels of inbred strains and a greater set of proteins will be needed to definitely relate variability in brain protein expression to function. We herein aimed to determine a “signaling protein phenotype” of the individual strains, thus contributing to information available at the behavioral, electrophysiological and mRNA level. Additionally, we attempted generation of a first signaling proteins reference database from hippocampi of individual mouse strains using an analytical tool for the concomitant determination of a large series of signaling structures by a protein-chemical method independent of antibody availability and specificity.

EXPERIMENTAL PROCEDURES Animals The subjects were FVB/NHim (FVB), C57BL/6JHim (C57), 129S2/ SvHim (129Sv), Balb/cHim (Balb) and Him:OF1 (OF) (n⫽10 for each strain) male, adult mice, approx. 20 weeks old. Animals were housed in standard transparent laboratory cages in a temperature-controlled colony room (22⫾1 °C). They were maintained on a 12 h artificial light/dark cycle (with lights on at 6:00 A.M.) and provided with food and water ad libitum. Mice were killed by neck dislocation and hippocampi were dissected. Tissue samples were immediately frozen in liquid nitrogen and stored at ⫺80 °C until used for analysis. All animal experiments were carried out in accordance with the European Community Council Directive (96/ 609/EEC) on animal welfare and approved by the local animal committee (confirmation number: LF1-TVG-17/002-2004). All efforts were made to minimize animal suffering and the number of animals used.

Sample preparation Hippocampal tissue was powdered and resuspended in 1.0 ml of sample buffer consisting of 7 M urea (Merck, Darmstadt, Germany), 2 M thiourea (Sigma, St. Louis, MO, USA), 4% CHAPS (3-[(3cholamidopropyl) dimethylammonio]-1-propane-sulfonate) (Sigma), 65 mM DTT (1,4-dithioerythritol; Merck), 1 mM EDTA (Merck), protease inhibitors completeâ (Roche, Basel, Switzerland) and 1 mM phenylmethylsulfonyl chloride. The suspension was sonicated for approximately 15 s. After homogenization samples were left at room temperature for 1 h and centrifuged at 14,000 r.p.m. for 1 h. The supernatant was transferred into Ultrafree-4 centrifugal filter unit (Millipore, Bedford, MA, USA), for desalting and concentrating proteins. Protein content of the supernatant was aimed at determining by Bradford protein assay system (Bradford, 1976). The standard curve was generated using bovine serum albumin and absorbance was measured at 595 nm.

Two-dimensional gel electrophoresis (2-DE) Samples prepared from each mouse individual mouse (n⫽10 per strain) were subjected to 2-DE as described elsewhere (Weitzdoerfer et al., 2002). Seven hundred micrograms protein was applied on immobilized pH 3–10 nonlinear gradient strips at their basic and acidic ends. Focusing was started at 200 V and voltage was gradually increased to 8000 V over 31 h and then kept constant for a further 3 h (approximately 150,000 Vh totally). After the first dimension, strips (18 cm) were equilibrated 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. After equilibration, strips were loaded on 9 –16% gradient sodium dodecylsulfate polyacrylamide gels for second-dimensional separation. Gels (180⫻200⫻1.5 mm) were run at 40 mA per gel. Immediately after the second dimension run gels were fixed for 18 h in 50% methanol, containing 10% acetic acid, the gels were then stained with Colloidal Coomassie Blue (Novex, San Diego, CA, USA) for 12 h on a rocking shaker. Molecular masses were determined by running standard protein markers (Biorad Laboratories, Hercules, CA, USA) covering the range 10 –250 kDa. pI values 3–10 were used as given by the supplier of the immobilized pH gradient strips (Amersham Bioscience, Uppsala, Sweden). Excess dye was washed out from the gels with distilled water and the gels were scanned with ImageScanner (Amersham Bioscience). Electronic images of the gels were recorded using Adobe Photoshop (Adobe Systems Inc., San Jose, CA, USA) and Microsoft PowerPoint (Microsoft Corp., Redmond, WA, USA) software.

Matrix-assisted laser desorption ionization mass spectrometry Spots (3⫻384) each were randomly picked and previous work on hippocampal signaling proteins protein expression was respected (Shin et al., 2004, 2005; Fountoulakis et al., 2005). Spots were excised with a spot picker (PROTEINEER spTM, Bruker Daltonics, Bremen, Germany), placed into 96-well microtiter plates and in-gel digestion and sample preparation for MALDI analysis were performed by an automated procedure (PROTEINEER dpTM, Bruker Daltonics) (Suckau et al., 2003; Yang et al., 2004). Briefly, spots were excised and washed with 10 mM ammonium bicarbonate and 50% acetonitrile in 10 mM ammonium bicarbonate. After washing, gel plugs were shrunk by addition of acetonitrile and dried by blowing out the liquid through the pierced well bottom. The dried gel pieces were reswollen with 40 ng/␮l trypsin (Promega, Madison, WI, USA) 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 was performed with 10 ␮l of 1% TFA in 5 mM OGP. Extracted peptides were directly applied onto a target (AnchorChipTM, Bruker Dalton-

D. D. Pollak et al. / Neuroscience 138 (2006) 149 –158 ics) that was load with ␣-cyano-4-hydroxy-cinnamic acid (Bruker Daltonics) matrix thin layer. The mass spectrometer used in this work was an UltraflexTM TOF/TOF (Bruker Daltonics) operated in the reflector mode for matrix-assisted laser desorption/ionizationtime of flight (MALDI-TOF) peptide mass fingerprint (PMF) or LIFT mode for MALDI-TOF/TOF with a fully automated mode using the FlexControlTM software. An accelerating voltage of 25 kV was used for PMF. Calibration of the instrument was performed externally with [M⫹H]⫹ ions of angiotensin I, angiotensin II, substance P, bombesin, and adrenocorticotropic hormones (clip 1–17 and clip 18 –39). Each spectrum was produced by accumulating data from 200 consecutive laser shots. Those samples which were analyzed by PMF from MALDI-TOF were additionally analyzed using LIFT-TOF/TOF MS/MS from the same target. A maximum of three precursor ions per 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 lifted by 19 kV to high potential energy in the LIFT cell. After further acceleration of the fragment ions in the second ion source, their masses could be simultaneously analyzed in the reflector with high sensitivity. PMF and LIFT spectra were interpreted with the Mascot software (Matrix Science Ltd., London, UK). Database 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 0 missing cleavage sites for PMF and MS/MS tolerance of 0.5 Da and one missing cleavage site for MS/MS search were allowed and oxidation of methionine residues was considered. The probability score calculated by the software was basically used as criterion for correct identification. The algorithm used for determining the probability of a false positive match with a given mass spectrum is described elsewhere (Berndt et al., 1999).

Generation of a master gel of signaling proteins Signaling proteins were selected and a combined master gel was constructed from all groups (10 individual mice i.e. gels per strain, see Fig. 1).

Quantification Protein spots representing signaling proteins from all gels (10 per group; total n⫽50) were outlined (first automatically and then manually) and quantified using the Proteomweaver software (Definiens, Munich, Germany). The percentage of the volume of the spots representing a certain protein was determined in comparison with the total proteins present in the 2-DE gel (Langen et al., 1999).

Statistical analysis Values are expressed as means⫾standard deviation (S.D.) of percentage of the spot volume in each particular gel after subtraction of the background values. Between-group differences were analyzed using the Kruskal-Wallis rank sum test for strain effects. Proteins with significant effects were selected by adjusting the resulting P-values for multiple testing. GraphPad InStat version (GraphPad Software, San Diego, CA, USA) was used for all statistical analysis and the level of significance was set at P⬍0.05 at all instances.

RESULTS A total of 46 spots corresponding to 37 different signaling proteins in hippocampal 2-DE gels of five different mouse strains has been analyzed by MALDI-TOF/MALDI-TOFTOF and identified through database search. Statistical analysis revealed strain-dependent expression of 11 dif-

151

ferent signaling molecules, i.e. serine/threonine protein phosphatase 1, beta catalytic subunit (PP1-beta), serine/ threonine protein phosphatase 1, gamma catalytic subunit (PP1-gamma), serine/threonine protein phosphatase 2A (PP2A), 65 kDa regulatory subunit A, alpha isoform, large GTP binding protein Opa1 (Dynamin-like 120 kDa protein), guanine nucleotide-binding protein beta subunit 1, putative GTP-binding protein PTD004, GTP-binding protein Ran, guanine nucleotide-binding protein beta subunit 2-like 1 (RACK1), WASP-family protein member (WAVE-1 protein), voltage-dependent anion channel 2 and 14-3-3 protein gamma. These proteins were represented by single spots each (Table 1a) and a master gel of signaling proteins present in all five mouse strains was constructed (see Fig. 1). Serine/threonine protein phosphatase 1 (PP1) PP1 consists of three catalytic subunits (PP1-alpha, -beta and -gamma). PP1- beta and -gamma showed strain dependent regulation with highest level of the beta subunit in 129Sv and lowest levels in FVB (see Table 2a). In C57, PP1-beta was detectable in only three out of 10 animals (no statistical evaluation was carried out). The gamma form showed highest expression level in 129Sv and lowest levels in OF. Serine/threonine PP2A PP2A exists as a multisubunit enzyme complex and is expressed at high levels in the CNS (Strack et al., 1998). The enzyme complex consists of a 36 kDa catalytic subunit (PP2A-C) and a 65 kDa structural subunit (PP2A-A) forming a core enzyme, which then associates with a variable regulatory subunit (PP2A-B) to constitute the heterotrimeric PP2A holoenzyme (Mumby and Walter, 1993). Expression of PP2A-A was strain-dependent with highest expressional levels observed in 129Sv and lowest levels in FVB (see Table 2a). Large GTP binding protein OPA1 Large GTP binding protein OPA1 is a member of the dynamin GTPase family. Regulation of large GTP binding protein OPA1 in a strain-dependent manner was revealed. 129Sv Displayed highest levels of expression while expression was lowest in FVB (see Table 2a). Herein an expression form with a more acidic observed pI probably indicating phosphorylation (Table 1a) was quantified in all five groups. Guanine nucleotide-binding protein beta subunit 1 (GNB1) Guanine nucleotide-binding proteins (G proteins) are composed of three units, alpha, beta and gamma. Strain-dependent expression of GNB1 was detected. Levels of GNB1 were highest in Balb and lowest in FVB (see Table 2a). Putative GTP-binding protein PTD004 Putative GTP-binding protein PTD004 belongs to the GTP1/OBG family. As yet, the function of these proteins is uncertain. Highest PTD004 levels were found in 129Sv while expression of PTD004 was lowest in FVB (see Table 2a).

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Fig. 1. Two-dimensional map of mouse hippocampal proteins. Mouse hippocampal proteins were extracted and 700 ␮g were applied on an immobilized pH 3–10 non-linear gradient strip, followed by 9 –16% linear gradient polyacrylamide gel as described in Experimental Procedures. Gels were stained with Coomassie Blue, spots were analyzed by MALDI-MS/MS-MS and proteins were assigned using MASCOT software. SWISS-PROT accession numbers for protein identification are given.

GTP-binding protein Ran GTP-binding protein Ran belongs to the Ras family within the superfamily of small GTPases (Helmreich, 2004).

Strain-dependent Ran expression was revealed with highest expression in Balb whereas 129Sv displayed lowest levels of Ran expression (see Table 2a).

D. D. Pollak et al. / Neuroscience 138 (2006) 149 –158

153

Table 1. Identification and characterization of signaling proteins in hippocampus of C57 (C), 129Sv (S), Balb (B), FVB (F) and OF (O) mice Accession number 1a P62140 P63087 Q76MZ3 P58281 P62873 Q9CZ30 P62826 P63245 Q8R5H6 Q78MH6 P61982 1b P14211 I52465 P18872

Name

MS score

Serine/threonine protein phosphatase 1, beta catalytic subunit Serine/threonine protein phosphatase 1, gamma catalytic subunit Serine/threonine protein phosphatase 2A, 65 kDa regulatory subunit A, alpha isoform Large GTP binding protein Opal (Dynamin-like 120 kDa protein) Guanine nucleotide-binding protein beta subunit 1 Putative GTP-binding protein PTD004 GTP-binding protein Ran/TC4 GTPase Ran Guanine nucleotide-binding protein beta subunit 2-like 1 (RACK1) WASP-family protein member 1 (WAVE-1 protein) Voltage-dependent anion channel 2 14-3-3 Protein gamma

Calcireticulin Phospatidylinositol transfer protein alpha Guanine nucleotide-binding protein G(o), alpha subunit 1 P40124 Adenylyl cyclase-associated protein 1 (CAP 1) P50396 RAB GDP dissociation inhibitor alpha P62138 Phosphoprotein phosphatase 1, alpha catalytic chain (PP1-alpha) P62259 14-3-3 Protein epsilon P62259 14-3-3 Protein epsilon P62259 14-3-3 Protein epsilon P62714 Serine/threonine protein phosphatase 2A, beta-isoform P62879 Guanine nucleotide-binding protein beta subunit 2 P63085 Mitogen-activated protein kinase 1 P63101 14-3-3 Protein zeta/delta P63101 14-3-3 Protein zeta/delta P63101 14-3-3 Protein zeta/delta P63101 14-3-3 Protein zeta/delta Q08331 Calretinin Q60631 Growth factor receptor-bound protein 2 (GRB2 adapter protein) Q60931 Voltage-dependent anion-selective channel protein 3 Q61598 Rab GDP dissociation inhibitor beta-2 (Rab GDI beta-2) Q62048 Astrocytic phosphoprotein PEA-15 Q8BG73 SH3 domain-binding glutamic acid-rich-like protein 2 Q8BK60 Serine proteinase inhibitor Q8BN77 Protein tyrosine kinase 9-likemouse Q8BP10 Rho GDP-dissociation inhibitor 1 Q8VIN1 Phosphatidylethanolamine-binding protein (PEBP) Q9CQV8 14-3-3 Protein beta/alpha Q9CWS0 NG-dimethylarginine dimethylaminohydrolase 1 Q9D7X3 Dual specificity protein phosphatase 3 Q9WU78 Programmed cell death 6 interacting protein Q9Z2L0 Voltage-dependent anion channel 1 (VDAC-1) Q9Z2L0 Voltage-dependent anion channel 1 (VDAC-1) Q9Z2L0 Voltage-dependent anion channel 1 (VDAC-1) Q9Z2L0 Voltage-dependent anion channel 1 (VDAC-1)

MS/MS score

Match. pept.

Sequ. cov. %

108

15

55

99

16

139

MS/MS peptides

Theor. MW

Theor. pI

Observ. pI

37,187

5.84

5.8

49

37,669

6.13

6.1

22

48

66,079

5.00

5.1

114

25

29

111,783

7.18

6.1

70 146 80 69

12 14 10 7

48 51 41 41

37,377 44,729 24,579 31,398

5.60 7.64 7.01 7.72

5.6 8 7.5 4.7

87 103 70

14 11 12

29 46 39

61,772 31,733 28,325

5.94 7.44 4.80

6.2 7.5 4.5

101 148 87

43 79 49

11 18 14

27 47 30

1 2 2

47,995 31,893 39,953

4.33 5.97 5.34

4.2 6.2 5.3

101 241 107

55 88

11 23 14

25 52 47

1 2

51,875 50,522 38,229

7.30 4.05 5.94

7.5 5.1 5.7

14 14 17 13

47 58 60 58

29,341 29,341 29,341 35,575

4.63 4.63 4.63 5.21

4 4.2 4.8 5.3

15 14 11 13 10 14 12 13

42 36 38 49 38 57 45 41

37,331 41,276 27,771 27,771 27,879 27,879 31,373 25,238

5.60 6.50 4.73 4.73 4.72 4.72 4.94 5.89

5.6 7.5 4.5 4.6 4.9 5.1 4.8 5.8

61

8

36

30,753

8.96

8.2

254

35

74

51,018

5.93

5.9

9 5 11 8 14 10 10 16 10 20 14 12 14 13

36 58 29 40 56 60 39 51 59 31 59 58 62 69

15,012 12,255 42,618 39,516 22,948 20,857 27,955 31,250 20,472 96,010 30,851 30,851 30,851 30,851

4.94 5.49 5.08 6.24 5.20 5.19 4.77 5.64 6.07 6.15 8.63 8.63 8.63 8.63

4.4 5.8 6 7.7 5.1 4.4 4.5 5.6 5.8 6.2 7.6 8.2 8.3 8.5

81 78 110 97 119 97 64 93 93 95 63 67

99 65 66 71 101 70 67 100 77 91 110 101 112 114

45

43

70

49

2

1

4

1

1a Contains proteins showing strain-dependent expression (see Table 2a); proteins with comparable expression levels among strains (see Table 2b) are listed in 1b. Accession number was retrieved from SWISS-PROT database (http://www.expasy.org/sprot/). Theoretical pI/molecular weight (Da) was predicted (http://www.expasy.org/cgi-bin/pi_tool). Observed pI refers to pI observed in master gel. Score is ⫺10*Log (P), where P is the probability that the observed match is a random event (MASCOT, http://www.matrixscience.com).

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Table 2. Expression levels of signaling proteins in hippocampus of 129Sv, C57, Balb, FVB and OF mice Accession number 2a P62140 P63087 Q76MZ3 P58281 P62873 Q9CZ30 P62826 P63245 Q8R5H6 Q78MH6 P61923 2b P14211 I52465 P18872 P40124 P50396 P62138 P62259 P62259 P62259 P62714 P62879 P63085 P63101 P63101 P63101 P63101 Q08331 Q60631 Q60931 Q61598 Q62048 Q8BG73 Q8BK60 Q8BN77 Q8BPI0 Q8VIN1 Q9CQV8 Q9CWS0 Q9D7X3 Q9WU78 Q9Z2L0 Q9Z2L0 Q9Z2L0 Q9Z2L0 Q9Z2L0

Name

Balb (n⫽10)

C57 (n⫽10)

FVB (n⫽10)

129Sv (n⫽10)

OF (n⫽10)

Serine/threonine protein phosphatase 1, beta catalytic subunit Serine/threonine protein phosphatase 1, gamma catalytic subunit Serine/threonine protein phosphatase 2A, 65 kDa regulatory subunit A, alpha isoform Large GTP binding protein opal (Dynamin-like 120 kDa protein) Guanine nucleotide-binding protein beta subunit 1 Putative GTP-binding protein PTD004 GTP-binding protein Ran/TC4 GTPase Ran Guanine nucleotide-binding protein beta subunit 2-like 1 (RACK1) WASP-family protein member 1 (WAVE-1 protein) Voltage-dependent anion channel 2 14-3-3 Protein gamma

0.13⫾0.05 0.06⫾0.03 0.29⫾0.10

n.d. 0.06⫾0.03 0.28⫾0.18

0.12⫾0.05 0.05⫾0.01 0.23⫾0.11

0.29⫾0.10 0.10⫾0.05 0.55⫾0.26

0.17⫾0.07 0.04⫾0.01 0.38⫾0.17

0.03⫾0.01 1.32⫾0.26 0.09⫾0.04 1.09⫾0.25 0.26⫾0.14

0.05⫾0.03 1.27⫾0.13 0.08⫾0.04 0.93⫾0.17 0.25⫾0.11

0.07⫾0.04 0.81⫾0.11 0.07⫾0.03 0.75⫾0.25 0.64⫾0.14

0.08⫾0.03 1.21⫾0.31 0.15⫾0.06 0.55⫾0.22 0.29⫾0.15

0.04⫾0.02 0.99⫾0.25 0.10⫾0.03 0.73⫾0.21 0.43⫾0.21

0.04⫾0.01 0.82⫾0.21 0.91⫾0.28

0.07⫾0.03 1.02⫾0.11 0.94⫾0.48

0.08⫾0.05 1.19⫾0.21 0.69⫾0.24

0.14⫾0.07 0.99⫾0.08 1.62⫾0.75

0.06⫾0.01 0.81⫾0.15 0.98⫾0.15

Calcireticulin Phospatidylinositol transfer protein alpha Guanine nucleotide-binding protein G(o), alpha subunit 1 Adenylyl cyclase-associated protein 1 (CAP 1) RAB GDP dissociation inhibitor alpha Phosphoprotein phosphatase alpha catalytic chain 14-3-3 Protein epsilon 14-3-3 Protein epsilon 14-3-3 Protein epsilon Serine/threonine protein phosphatase 2A, beta-isoform Guanine nucleotide-binding protein beta subunit 2 Mitogen-activated protein kinase 1 14-3-3 Protein zeta/delta 14-3-3 Protein zeta/delta 14-3-3 Protein zeta/delta 14-3-3 Protein zeta/delta Calretinin Growth factor receptor-bound protein 2 (GRB2 adapter protein) Voltage-dependent anion-selective channel protein 3 Rab GDP dissociation inhibitor beta-2 (Rab GDI beta-2) Astrocytic phosphoprotein PEA-15 SH3 domain-binding glutamic acid-rich-like protein 2 Serine proteinase inhibitor Protein tyrosine kinase 9-likemouse Rho GDP-dissociation inhibitor 1 Phosphatidylethanolamine-binding protein (PEBP) 14-3-3 Protein beta/alpha NG-dimethylarginine dimethylaminohydrolase 1 Dual specificity protein phosphatase 3 Programmed cell death 6 interacting protein Voltage-dependent anion channel 1 (VDAC-1) Voltage-dependent anion channel 1 (VDAC-1) Voltage-dependent anion channel 1 (VDAC-1) Voltage-dependent anion channel 1 (VDAC-1) Voltage-dependent anion channel 1 (VDAC-1)

0.75⫾0.19 0.35⫾0.05 0.44⫾0.21 0.17⫾0.04 0.55⫾0.25 0.18⫾0.08 0.62⫾0.26 0.03⫾0.01 0.68⫾0.27 0.26⫾0.12 1.14⫾0.3 0.15⫾0.04 1.72⫾0.6 1.23⫾0.48 1.35⫾0.66 0.55⫾0.21 1.22⫾0.43 0.17⫾0.04 0.33⫾0.08 0.3⫾0.14 0.31⫾0.13 0.23⫾0.07 0.06⫾0.02 0.23⫾0.14 0.33⫾0.13 0.35⫾0.04 0.65⫾0.35 0.44⫾0.13 0.13⫾0.04 0.28⫾0.1 0.35⫾0.14 0.65⫾0.32 1.08⫾0.33 1.12⫾0.5 0.44⫾0.21

0.67⫾0.27 0.33⫾0.07 0.46⫾0.18 1.13⫾0.55 0.38⫾0.12 0.15⫾0.05 0.54⫾0.18 0.05⫾0.02 0.71⫾0.28 0.26⫾0.09 1.37⫾0.33 0.16⫾0.08 1.37⫾0.57 1.17⫾0.4 1.42⫾0.44 0.62⫾0.3 1.31⫾0.33 0.15⫾0.04 0.34⫾0.15 0.26⫾0.1 0.42⫾0.22 0.22⫾0.05 0.05⫾0.02 0.18⫾0.06 0.31⫾0.13 0.35⫾0.05 0.61⫾0.24 0.49⫾0.21 0.15⫾0.04 0.21⫾0.08 0.52⫾0.17 0.69⫾0.27 0.99⫾0.20 1.23⫾0.48 0.57⫾0.24

0.72⫾0.33 0.37⫾0.11 0.49⫾0.2 0.18⫾0.09 0.52⫾0.11 0.24⫾0.07 0.48⫾0.07 0.06⫾0.03 0.73⫾0.17 0.28⫾0.1 0.84⫾0.17 0.17⫾0.04 0.17⫾0.04 1.13⫾0.55 1.22⫾0.33 0.49⫾0.21 1.32⫾0.37 0.18⫾0.05 0.29⫾0.12 0.26⫾0.13 0.37⫾0.17 0.14⫾0.07 0.05⫾0.02 0.08⫾0.04 0.26⫾0.12 0.44⫾0.13 0.95⫾0.33 0.53⫾0.11 0.1⫾0.04 0.26⫾0.09 0.48⫾0.18 0.72⫾0.28 1.07⫾0.33 1.21⫾0.54 0.53⫾0.33

0.84⫾0.21 0.35⫾0.04 0.47⫾0.11 1.14⫾0.3 0.54⫾0.18 0.20⫾0.1 0.67⫾0.27 0.04⫾0.03 0.67⫾0.35 0.26⫾0.11 1.17⫾0.4 0.16⫾0.06 0.16⫾0.03 1.08⫾0.33 1.45⫾0.66 0.56⫾0.23 1.25⫾0.45 0.17⫾0.04 0.33⫾0.12 0.23⫾0.12 0.26⫾0.12 0.15⫾0.08 0.07⫾0.03 0.19⫾0.10 0.31⫾0.11 0.38⫾0.12 0.65⫾0.32 0.55⫾0.22 0.12⫾0.07 0.22⫾0.08 0.38⫾0.17 0.66⫾0.31 0.95⫾0.26 1.14⫾0.33 0.48⫾0.22

0.78⫾0.28 0.36⫾0.05 0.45⫾0.13 0.15⫾0.04 0.53⫾0.23 0.18⫾0.09 0.55⫾0.25 0.06⫾0.02 0.66⫾0.32 0.24⫾0.12 1.04⫾0.36 0.23⫾0.11 0.15⫾0.04 1.34⫾0.54 1.03⫾0.46 0.6⫾0.18 1.42⫾0.46 0.16⫾0.03 0.36⫾0.16 0.18⫾0.09 0.24⫾0.12 0.21⫾0.14 0.06⫾0.02 0.19⫾0.1 0.31⫾0.12 0.33⫾0.07 0.85⫾0.32 0.47⫾0.21 0.15⫾0.04 0.26⫾0.07 0.43⫾0.19 0.76⫾0.07 0.89⫾0.19 1.13⫾0.55 0.51⫾0.19

Proteins were analyzed by 2-DE and identified by MALDI-TOF. Proteins showing strain-dependent expression are listed in 2a, proteins with comparable expressional levels in 2b. Relative protein expression resulting from software-assisted quantification are given (mean⫾SD) (n.d., not detectable).

RACK1 RACK1, located in the small ribosomal subunit, is a scaffold protein that is able to interact simultaneously with several signaling molecules. It binds to protein kinases and membranebound receptors in a regulated fashion (Brandon et al., 2002).

Regulation of RACK1 expression was found to be straindependent. FVB showed highest levels of RACK1 while RACK one expression was lowest in C57 (see Table 2a). Herein an acidic expression form with an observed pI much lower than the predicted one (see Table 1a) was quantified in all groups.

D. D. Pollak et al. / Neuroscience 138 (2006) 149 –158

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Table 3. Statistical analysis of signaling protein expression in hippocampus of C57 (C), 129Sv (S), Balb (B), FVB (F) and OF (O) mice Accession number

Main effect, strain effect

Strain-wise comparison S:B

S:C

S:F

S:O

B:C

B:F

B:O

C:F

C:O

F:O

P61923 P62826 P62873 P58281 P62140 P63087 Q76MZ3 Q9CZ30 P63245 Q78MH6 Q8R5H6

P⬍0.018 P⬍0.0024 P⬍0.0044 P⬍0.04 P⬍0.0015 P⬍0.023 P⬍0.018 P⬍0.032 P⬍0.0006 P⬍0.0012 P⬍0.0013

P⬍0.05 P⬍0.01 n.s. P⬍0.05 P⬍0.01 n.s. n.s. n.s. n.s. n.s. P⬍0.01

n.s. P⬍0.05 n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. P⬍0.05

P⬍0.05 n.s. P⬍0.05 n.s. P⬍0.01 n.s. P⬍0.05 P⬍0.05 P⬍0.01 n.s. n.s.

n.s. n.s. n.s. n.s. P⬍0.05 P⬍0.05 n.s. n.s. n.s. n.s. P⬍0.01

n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.

n.s. n.s. P⬍0.05 n.s. n.s. n.s. n.s. n.s. P⬍0.01 P⬍0.01 n.s.

n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.

n.s. n.s. P⬍0.05 n.s. n.s. n.s. n.s. n.s. P⬍0.01 n.s. n.s.

n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.

n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. P⬍0.01 n.s.

Statistical analysis was carried out using Kruskal-Wallis rank sum test for strain effects. Proteins with significant effects were selected by adjusting the resulting P-values for multiple testing. (n.s. is not significant).

WAVE-1 protein The Scar/WAVE family of scaffolding proteins organize molecular networks that relay signals from the GTPase Rac to the actin cytoskeleton. The WAVE-1 isoform is a brain-specific protein expressed in a variety of areas including the hippocampus. Strain-dependent expression of WAVE-1 protein was detected with highest levels in 129Sv and lowest levels in Balb (see Table 2a). Voltage-dependent anion channel (VDAC) 2 VDAC is present in three forms (VDAC 1, VDAC 2, and VDAC 3) in the mammalian brain. Strain-dependent expression of VDAC 2 has highest levels in 129Sv and lowest in FVB (see Table 2a). 14-3-3 Protein gamma 14-3-3 protein gamma belongs to a family of highly conserved adaptor proteins, which bind to the phosphoserinecontaining motifs of the target protein and is mainly localized to the synapses and neuronal cytoplasm. Regulation of 14-3-3 protein gamma expression was found to be strain-dependent. 129Sv showed highest levels of 14-3-3 protein gamma while expression was lowest in FVB (see Table 2a). Proteins with comparable expressional levels No effect of strain and comparable signaling protein levels was observed for the other proteins listed in Table 1b. Thirty-five protein spots representing 26 different proteins with comparable expressional levels were observed. Strain effect Statistical analysis of signaling proteins expression in the individual mouse strains is shown in Table 3.

DISCUSSION The hippocampus has been a major target of analysis in the search for molecular and cellular correlates to plastic alter-

ations thought to underlie persistence in synaptic strength required for NIS. The responsible signaling mechanisms therefore comprise a host of molecules and sophisticated interactive networks. As such, only an experimental approach suiting the complexity of the biological conditions is expected to provide insight into the underlying regulatory principles and we decided to carry out proteomic analysis to evaluate variability in the expression of signaling proteins among different mouse strains. And indeed, this study provides evidence that several key signaling proteins appear to be regulated in a strain-dependent manner in the mouse hippocampus. It is intriguing to see that levels of significantly and differently expressed signaling proteins in individual mouse strains are varying up to two-fold (Table 2a). The beta catalytic subunit of PP1 was not even detectable in more than three out of 10 samples from C57. We are aware of the fact that lower levels in individual mouse strains do not necessarily reflect altered function of the signaling cascades and that polymorphisms as well as posttranslational modifications may have led to different expression and we were only detecting signaling proteins with specific pIs at a certain position on the gel. Moreover, we cannot claim that the individual signaling proteins observed are the major functional structures of the corresponding cascades and we are only able to analyze a limited amount of high abundance (i.e. positive Coomassie-stained spots) proteins. However, proteins found to be expressed in a strain-dependent manner herein belong to protein groups with assigned functional properties for neuronal information processing and storage. Serine/threonine protein phosphatases (PPs) (PP1, PP2A) Protein phosphorylation is thought as one of the key signaling process involved in NIS that operates through a tight balance between the action of protein kinases and PPs. Four major PPs (PP1, PP2A, PP2B, PP2C), able to specifically remove phosphate groups from serine and threonine residues and are highly abundant in the brain where, together with protein kinases, they contribute to the control

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of synaptic plasticity and memory. It has become increasingly clear that the actions of the individual phosphatases regulate NIS in a highly specific and distinct manner (reviewed in Munton et al., 2004). Elegant gene-targeting approaches have proven a functional in vivo importance of PPs in the context of learning and memory (Malleret et al., 2001; Gotz and Schild, 2003). Herein we show that the expression of several PP subunits is regulated in a straindependent manner in the mouse hippocampus. It would be tempting to associate strain-dependent PP-expression with strain-specific cognitive performance (Crawley et al., 1997) and future studies assessing PP-expression and cognitive performance in parallel may provide further information to this end. Guanine nucleotide-binding/and -associated proteins (GNB1, large GTP-binding protein Opa1, putative GTP-binding protein PTD004, GTP-binding protein Ran) G-protein pathways are involved in the regulation of NIS through various signaling cascades. Through the generation of cAMP (cyclic AMP) acting through PKA (protein kinase A) short-term effects on channel functions can be produced. Activation of small GTP-ases and MAPK may regulate gene expression and produce long-term effects through regulation of the transcriptional machinery (Neves et al., 2002). We are able to provide evidence that several components of G-protein-related pathways are differentially expressed among various wild-type inbred mouse strains. As such, evidence for genetic regulation of the expression of individual proteins related to G-protein signaling is provided and can be linked to studies at the DNA and messenger level. Kitanaka et al. (2003) identified eight single nucleotide polymorphisms, three insertions and one deletion when comparing the sequence from 129Sv and C57Bl/6 strains at the GNB1 gene locus of which the corresponding proteins have been found to be regulated in a strain-dependent manner herein. Furthermore Fernandes et al. (2004) showed strain-dependent expression of GNB1 at the messenger level when carrying out gene expression profiling of hippocampal tissue among eight different mouse strains. Ion channels (VDAC-2) Opening of voltage-gated ion channels located in the neuronal membrane is mandatory when an excitatory synapse becomes activated. In view of this, regulations of ionic conductances is discussed as one of the mechanisms modulating the propagation of neuronal information (Daoudal and Debanne, 2003) on the long-term. Here we show strain-dependent regulation of VDAC in the mouse hippocampus. Considering that VDAC-deficient mice present with disrupted spatial learning and fear conditioning we propose that differential expression of VDAC may be related to the variability in cognitive performance described for the mouse strains examined herein (Crawley et al., 1997) although further studies will be needed to directly associate protein expression and neurophenotype.

Miscellaneous RACK1. RACK1 has been proposed to function primarily as a “shuttle or anchoring protein,” facilitating the targeting of PKC isoforms to their substrates. Brandon et al. (2002) showed a role for RACK1 in modulating GABAA receptor, phosphorylation and the functional modulation by PKC-dependent cell signaling pathways. Given that GABAA receptors are the principal sites of fast synaptic inhibition in the brain and the widespread importance of PKC-dependent signaling pathways, from MAPK to CaMKII cascade, the relevance of RACK1 in the modulation of signaling cascades critical for NIS becomes evident. This implication is supported by findings of aberrant expression of RACK1 in fetal Down syndrome brain and its potential relevance for cognitive deficits observed in Down syndrome patients (Peyrl et al., 2002). 14-3-3 Protein gamma. 14-3-3 Proteins are a family of highly conserved molecules mainly localized to the synapses and neuronal cytoplasm. Originally known as adaptor proteins, they are now endowed with a growing series of potential functions and pathological relevance. Increased levels of 14-3-3 gamma have been detected in brains of patients with Alzheimer’s disease and Down syndrome (Fountoulakis et al., 1999) whereas 14-3-3 was reduced in fetal Down syndrome brain (Peyrl et al., 2002). Alterations of 14-3-3 protein gamma expression may lead to or represent impaired neuronal differentiation, aberrant signaling and synaptic plasticity. This study was designed to investigate strain-dependent regulation of signaling proteins in the mouse hippocampus. We focused on characterization and quantification of structures which we have previously identified from mouse hippocampal tissue and assigned to the respective function (Shin et al., 2004, 2005). These first findings are of relevance for future design of studies on signaling proteins in individual mouse strains and show that data from individual mouse strains cannot be simply extrapolated and compared. Signaling proteins described herein have been observed in most brain protein reference databases (Shin et al., 2005; Yang et al., 2004; Fountoulakis et al., 2005) and are fairly identified. The limited amount of identified signaling proteins analyzed has to be significantly extended to warrant protein chemical determination of the many known signaling proteins by proteomic techniques rather than by immunochemical methods dependent on antibody availability and specificity. Although we did not correlate differences in protein expression observed herein with results obtained by other methods, unambiguous identification by MALDI analysis (mean sequence coverage over 46%, see Table 1) and reliable software-assisted quantification warrant fair interpretation of our results. Moreover, we have previously analyzed strain-dependent expression of synaptic plasticity-related proteins in a parallel experiment and were also able to reveal an effect of strain on hippocampal protein expression by Western blotting (Pollak et al., 2005).

D. D. Pollak et al. / Neuroscience 138 (2006) 149 –158

These results hold considerable promise that further proteomic studies of brain proteins will reveal strain-dependent regulation of more proteins involved in other processes contributing to the neurobehavioral phenotypes of the individual strains. We are aware of the fact that neither all genetic differences that influence a phenotype will manifest as protein expression level differences nor will all expressional differences necessarily be paralleled by behavioral variation. Enhanced or reduced expression of a specific protein may also result from polymorphisms in regulatory sequences of the respective genes or strain-dependent expression of other genes/protein strains relevant for translation or posttranslational processing or some of the protein expression differences found may have been fixed by chance during inbreeding. Furthermore, since brains have not been perfused with saline prior to dissection, hippocampal homogenates may have been contaminated with proteins from blood cells. Moreover, we cannot claim that the individual proteins observed are the major functional structures of the corresponding pathways since we are only able to analyze and identify a limited amount of high abundance (i.e. positive Coomassie-stained spots) proteins. Nevertheless, this study provides evidence for protein biochemical characteristics inherent to the individual strains and presents a first attempt to link protein expression measurements and phenotypic observations to a manageable number of candidate proteins. As such, these protein expression experiments may provide an indication of the genetic elements that, through the expressional level of the translated protein, contribute to the phenotypic differences between the selected strains. When considering the genealogies of the inbred mouse strains used, it is worth noting that C57BL, 129, and BALB/c mice share a common origin but all split into independent lines at the beginning of the last century whereas FVB/N mice belong to the apparently separate group of Swiss mice (Beck et al., 2000). Interestingly, FVB/N mice presented as outliner at the higher (two spots) or lower end (five spots) in seven out of the 11 spots showing strain-dependent regulation. However, without additional information, it is untrustworthy to ascribe a definitive role to any particular differentially expressed protein in contributing to the individual neurophentotypes. Data presented herein clearly show that expression of signaling proteins representing key components of signaling pathways implicated in NIS, is affected by the mouse strain examined. We thus have generated a “signaling protein phenotype” of individual strains hereby complementing previous studies reporting strain-dependent differences in hippocampal LTP (Gerlai, 2002) and gene expression (Fernandes et al., 2004) as well as data presenting straindependent regulation of individual proteins (Bachtell et al., 2002), (Mosquera et al., 2003). In view of this, the wellknown implications for the genetic background of genetically modified mice with regard to behavioral analysis (Lathe, 1996) are extended to the biochemical level. As such, basal expressional level of a candidate gene in the background strains should be considered when planning

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gene-targeting/transgenic experiments in order to ensure reliable differentiation between wild-type and genetically engineered animals. Considering the abovementioned importance of the proteins analyzed herein for signaling pathways involved in NIS, it is astonishing that, to our best knowledge, potential strain-dependent expressional regulation has been investigated so far only at the nucleic acid level (Fernandes et al., 2004; Kitanaka et al., 2003). Our results for the first time show significant and remarkable differences in the expression of signaling proteins between individual mouse strains. By treating protein expression as a phenotype, we have shown significant genetic variation in brain protein expression. These strainspecific differences must be considered when analyzing the functions and interactions of a particular protein of interest in either of the strains investigated and are thus of relevance for future design of studies on signaling proteins in the mouse hippocampus. Acknowledgments—The excellent technical contribution of Maureen B. Felizardo-Cabatic is highly appreciated.

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(Accepted 2 November 2005) (Available online 19 December 2005)