Fluctuations of hippocampal neuronal protein levels over the estrous cycle in the rat

Available online at www.sciencedirect.com

Neurochemistry International 52 (2008) 1002–1011 www.elsevier.com/locate/neuint

Fluctuations of hippocampal neuronal protein levels over the estrous cycle in the rat Wei-Fei Diao a, Wei-Qiang Chen a, Harald Ho¨ger b, Arnold Pollak a, Gert Lubec a,* a b

Department of Pediatrics, Medical University of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria Institute for Animal Genetics, Medical University of Vienna, Brauhausgasse 34, A-2325 Himberg, Austria Received 20 August 2007; received in revised form 10 October 2007; accepted 15 October 2007 Available online 22 October 2007

Abstract Hippocampal function is known to be estrous-cycle-dependent but information on estrous-cycle-dependent protein expression is limited. It was therefore the aim to study protein levels of the neuronal network over the estrous cycle in the hippocampus of female rats and in males showing protein chemical neuroanatomy in this area. Female and male OFA Sprague–Dawley rats were used and females were grouped to proestrous, estrous, metestrous and diestrous by using vaginal smears. Hippocampal tissue was taken, proteins extracted, run on two-dimensional gel electrophoresis and proteins were identified by mass spectrometry methods (MALDI-TOF-TOF and nano-LC-ESI-MS/MS). Spot volumes were quantified with specific software. A Synapsin-1 expression form was differentially regulated between proestrous and diestrous, a Synapsin IIa expression form was differentially regulated between proestrous and metestrous, the sum of ERC-2 proteins organizing the cytomatrix at the nerve terminals active zone was showing sex-dependent levels in the proestrous phase and Neurofilament triplet L protein was differentially expressed between the estrous phase and males. The findings may represent estrous-cycle-dependent hippocampal synaptic function that has been shown already in terms of electrophysiology and neuroanatomy. Neurofilament changes over the estrous cycle may reflect endoskeleton changes over the estrous cycle. We learn from this study, although increasing complexity of protein knowledge, that the estrous cycle and not only the sex per se has to be taken into account for design of future studies and interpretation of previous work at the protein level. # 2007 Elsevier Ltd. All rights reserved. Keywords: Neuronal; Estrous cycle; Hippocampus; Mass spectrometry; Synapse

Hippocampus biochemistry, morphology and function vary across the female estrous cycle (EC) as well as between sexes. Proestrous presents with increased dendritic spine density and numbers as revealed by histological investigations (Cooke and Woolley, 2005; Woolley and McEwen, 1992). Estrogen has beneficial effects on hippocampal synaptic plasticity in vivo and in vitro (Woolley, 1998), and increases performance in different hippocampus-dependent tests for cognitive function (Korol and Kolo, 2002; Sandstrom and Williams, 2001; Wood et al., 2001), an observation in agreement with human verbal and spatial performance (Halpern and Tan, 2001). However, controversial results were obtained in different behavioral tasks (Berry et al., 1997; Stackman et al., 1997; Warren and Juraska, 1997) and may be the consequence of different molecular

* Corresponding author. Tel.: +43 1 40 400 3215; fax: +43 1 40 400 6065. E-mail address: [email protected] (G. Lubec). 0197-0186/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2007.10.013

mechanisms underlying different types of tasks (Mizuno and Giese, 2005) or due to concomitant changes of estrogen, progesterone and other sex hormones over the EC (Chesler and Juraska, 2000). Effects of gonadal hormones on expression of genes involved in synaptic modeling/remodeling have been reported at the transcriptional level (Devidze et al., 2005) over the estrous cycle (Adams et al., 2001; Crispino et al., 1999). Synaptic communication requires an efficient synapticvesicle cycle, including vesicle biogenesis, transport and interaction with the cytoskeleton, uptake and storage of neurotransmitters, and regulated endocytosis and exocytosis (Jahn, 2004; Sudhof, 2004). These processes are governed by vesicle-associated or integral membrane proteins. Numerous presynaptic proteins have been identified (for reviews: Royle and Lagnado, 2003; Sudhof, 2000) including those involved in vesicle biogenesis, vesicle fusion. Proper functioning of the nervous system requires precise control of neurotransmitter release and since synaptic vesicles are loaded with neuro-

W.-F. Diao et al. / Neurochemistry International 52 (2008) 1002–1011

transmitters, much endeavor has been made to describe the effect of some individual synaptic proteins on neurotransmission and hence synaptic plasticity. Changes in synaptic interconnections among neurons, such as regulation of number of synaptic boutons and pool size of synaptic vesicles, are established to characterize the mechanism of synaptic plasticity. It has been well documented that expression alterations of various presynaptic proteins involved in synaptic function and homeostasis occur in several neurological and psychiatric disorders accompanied with cognitive deficits (Mirnics et al., 2001; Mirnics et al., 2000; Virmani et al., 2005). To the best of our knowledge systematic studies on protein profiling over the EC of neuronal network-related proteins have not been published so far thus forming the rationale for the current study. It was therefore the aim of this work to address potential fluctuations of these corresponding hippocampal proteins over the EC and to search for sex-related changes. 1. Experimental procedures 1.1. Materials Immobilized pH-gradient (IPG) strips and IPG buffers were purchased from Amersham Biosciences, part of GE Healthcare. Acrylamide/piperazine-diacrylamide (PDA) and the other reagents for the polyacrylamide gel preparation were purchased from Bio-Rad Laboratories (Hercules, CA, USA). CHAPS was obtained from Roche Diagnostics (Mannheim, Germany), urea from AppliChem (Darmstadt, Germany), thiourea from Fluka (Buchs, Switzerland), 1,4dithioerythritol (DTE) and EDTA from Merck (Darmstadt, Germany), and tributylphosphine (TBP) from Pierce Biotechnology (Rockford, IL, USA).

1.2. Animals Eight to 10 weeks old male (n = 10) and female (n = 50) OFA Sprague– Dawley rats were used. They were bred and maintained in an animal barrier facility in Makrolon type IV cages with woodchip bedding. A standard rodent diet and water from automatic valves were available ad libitum. Lights were on from 5:00 to 19:00. State of the estrous cycle in females was detected by daily vaginal smears, taken between 9:00 and 11:00. A small cotton tip moistened with water was inserted into the vagina and smeared on a microscopic slide. After fixation with methanol and staining with Giemsa, stain cells were examined under a microscope. By the appearance of vaginal smears the female rats were assigned to five phases (Baker, 1979; Maeda et al., 2000), proestrous (PE), estrous (E), early metestrous (ME1), late metestrous (ME2) and diestrous (DE). Only rats showing at least two regular 4–5 days cycles were used for the study. After euthanasia with CO2 dissection and removal of brain was done between 10:00 and 12:00. Hippocampal tissues were dissected, frozen immediately in liquid nitrogen and stored at 80 8C until use and the freezing chain was never interrupted. Experiments were performed according to the EU guidelines for animal experiments.

1.3. Sample preparation Individual rat hippocampus was powderised in liquid nitrogen and suspended in 2 ml sample buffer and processed as given previously (Myung and Lubec, 2006). The protein content of the supernatant was determined by the Bradford assay (Bradford, 1976).

1.4. Two-dimensional gel electrophoresis (2-DE) 2-DE was performed essentially as reported (Yang et al., 2004). Samples of 750 mg protein were applied on immobilized pH 3–10 nonlinear gradient strips.

1003

Focusing started at 200 V and the voltage was gradually increased to 8000 V at 4 V/min and kept constant for a further 3 h (approximately 150,000 V h totally). The second-dimensional separation was performed on 10–16% gradient SDSPAGE. After protein fixation for 12 h in 50% methanol and 10% acetic acid, the gels were stained with colloidal Coomassie blue (Novex, San Diego, CA, USA) for 8 h and excess of dye was washed out from the gels with distilled water. Molecular masses were determined by running standard protein markers (BioRad Laboratories, Hercules, CA, USA), covering the range of 10–250 kDa. Isoelectric point values were used as given by the supplier of the immobilized pH gradient strips.

1.5. In-gel digestion Spots were excised with a spot picker (PROTEINEER spTM, Bruker Daltonics, Leipzig, Germany), placed into 96-well microtiter plates (Bruker Daltonics, Leipzig, Germany) and in-gel digestion and sample preparation for MALDI analysis were performed by an automated procedure (PROTEINEER dpTM, Bruker Daltonics) (Gulesserian et al., 2007).

1.6. MALDI-TOF-TOF mass spectrometry and data processing A target (AnchorChipTM, Bruker Daltonics, Bremen, Germany) was wiped using paper tissues in sequence with acetone and N-heptane, followed by ultrasonicated in isopropanol and then in HPLC grade water, and dried in air. Four microliter extracted peptides were directly applied onto the target that was loaded with a-cyano-4-hydroxy-cinnamic acid (Bruker Daltonics, Bremen, Germany) matrix thinlayer. The mass spectrometer used in this work was an UltraflexTM TOF/TOF (Bruker Daltonics, Bremen, Germany) operated in the reflector for MALDI-TOF peptide mass fingerprint (PMF) or LIFT mode for MALDI-TOF-TOF MS/MS with a fully automated mode using the FlexControlTM software. Samples were analyzed by one PMF from MALDI-TOF, followed by additional LIFT-TOF/TOF MS/MS analysis of three peptides. Data were accumulated from 200 consecutive laser shots to produce PMF and MS/MS spectra. Peptide standard was used as external calibration. The m/z range is 700–4000 for PMF and 40–2560 for MS/MS. PMF and MS/MS spectra were interpreted primarily with the FlexAnalysisTM software (Bruker Daltonics, Bremen, Germany). Signal-to-noise ratio threshold was set as three. Autoproteolysis products of trypsin were used as internal calibration. Database searches, through the MASCOT 2.1 (Matrix Science, London, UK) against MSDB 20051115 database, using combined one PMF and three MS/MS datasets were performed via BioTools 2.3 software (Bruker Daltonics, Bremen, Germany). Species were limited to rodents. Enzyme was limited to trypsin with one maximum missing cleavage site. A mass tolerance of 25 ppm for PMF, MS/MS tolerance of 0.5 Da, fix modification of carbamidomethyl(C), and variable modification of oxidation of methionine and phosphorylation (Tyr, Thr, and Ser) were considered. A randomized MSDB database (http://www. matrixscience.com/help/decoy_help.html) was searched simultaneously and false positive results were omitted. Protein identification returned from MASCOT were manually examined and filtered to create a confirmed protein identification list. Positive protein identifications were based on a significant MOWSE score.

1.7. Analysis of peptides by nano-LC-ESI-MS/MS and data processing Spots that were not identified by MALDI-TOF-TOF were analyzed by a second mass spectrometrical approach (Chen et al., 2006). Database search was performed as above.

1.8. Quantification Protein spots representing neuronal proteins from all gels (10 per group; total n = 60) were outlined and matched, first automatically and then manually, and quantified using the Proteomweaver software (Definiens, Munich, Germany). The level of each protein spot volume was represented by its intensity in the 2-DE gel (Frischer et al., 2006).

1004

W.-F. Diao et al. / Neurochemistry International 52 (2008) 1002–1011

1.9. Statistics Statistical analysis was performed by using GraphPad Instat 3.05 (GraphPad Software, Inc., San Diego, CA, USA). Each protein spot volume and summarized levels of multiple spot proteins were input as raw data. Mean and standard deviation were calculated. Subsequently, one-way analysis of variance (ANOVA) was performed to find significantly regulated neuronal proteins. Only those proteins which had ANOVA P values less than 0.05 were performed subsequently with Tukey multiple comparison test to compare all pairs of groups and to correct for multiple testing. P values of Tukey multiple comparison post hoc test less than 0.01 (**) and less than 0.001(***) were considered as significant. P levels >0.01 and <0.05 (*) were considered as a trend but are not further discussed. The identification of all the differentially ‘‘expressed’’ proteins was re-confirmed by nano-ESI-LC-MS/MS and error tolerance search were carried out to detect modifications based on the definitions included at http://unimod.org/.

2. Results The estrous phases were characterized by the following different cell types as shown in Fig. 1: PE: the major population

are nucleated cells, sometimes mixed with cornified cells; E: cornified cells only; ME1 (late estrous/early metestrous): abundant caseous masses consisting of cornified cells, some nucleated cells may appear; ME2 (late metestrous/early diestrous): leukocytes mixed with various numbers of cornified cells; DE: leukocytes mixed with nucleated cells, some cornified cells may still be visible. The cellular morphology of vagina confirmed the estrous status of the rats involved in current study. Rat hippocampal proteins from six groups: male (M), PE; E; ME1; ME2; and DE were separated on two-dimensional gel electrophoresis and a combined map is shown in Fig. 2. One master gel from each group was identified with a success rate of more than 90% (normally 1152 spots were picked from one gel). Fifty six spots, representing 23 neuronal proteins, have been unequivocally identified by MALDI-TOFTOF and nano-LC-ESI-MS/MS (Fig. 2, supplementary Table 1). The identification results of these differentially expressed proteins are listed in Table 1.

Fig. 1. Microscopic identification of the different phases of the estrous cycle. Preestrous (PE), estrous (E), late estrous/early metestrous (ME1), early diestrous/late metestrous (ME2), and diestrous (DE) are each characterized by the number and morphology of the cells in vaginal smear. Magnification 400.

W.-F. Diao et al. / Neurochemistry International 52 (2008) 1002–1011

1005

Fig. 2. Representative two-dimensional map of rat hippocampal neuronal proteins. Hippocampal proteins of female rats in different estrous phases and male rats were extracted and 750 mg were applied on an immobilized pH 3–10 non-linear gradient strip, followed with 10–16% linear gradient polyacrylamide gel electrophoresis with Coomassie Blue staining. Spots were firstly identified by using MALDI-TOF-TOF and those unidentified spots were then applied to nano-LC-ESI-MS/MS analysis. UniProtKB accession numbers for protein identification are given and the accession numbers of significantly changed proteins are shown in white and the zoomed images are provided.

Quantification results as shown in Table 2 revealed EC- and sex-dependent levels of 8 neuronal proteins with ANOVA P value less than 0.05, including Synapsin-1, Synapsin IIa and IIb, Synaptotagmin-1, Synuclein beta, ERC protein-2, Brain acid soluble protein 1, Neurofilament triplet L protein. The full list of quantification results is shown in supplementary Table 2. Comparison between all pairs of

groups revealed no neuronal protein differences between estrous and early metestrous, estrous and diestrous, early metestrous and diestrous, late metestrous and diestrous. Synaptotagmin-I revealed an apparent molecular weight of 37 kDa and was observed at a pI of 6.0. This electrophoretic shift in mobility may be due to posttranslational modifications, to the presence of a splice variant or may be by chemical

1006

W.-F. Diao et al. / Neurochemistry International 52 (2008) 1002–1011

Table 1 Identification and characterization of differentially expressed neuronal proteins of rat hippocampus Protein name

Accession Number

Synapsin-1

P09951

Synapsin IIa

Q63537

spot number

MS score

Peps

MS%

MWt (KDa)

PIt

(1)

191

28

58

74.0

9.81

(2)

182

24

57

74.0

(3)

132

21

49

(4)

87

14

(5)

226

24

MS/MS score

MS/MS (%)

MS/MS peptide sequence

Posttranslational modifications

387

27

Oxidation: M343,M349

9.81

548

23

74.0

9.81

417

29

38

74.0

9.81

493

23

53

74.0

9.81

436

25

K.TYATAEPFIDAK.Y K.EMLSSTTYPVVVK.M R.VLLVIDEPHTDWAK.Y K.TNTGSAMLEQIAMSDR.Y K.VDNQHDFQDIASVVALTK.T K.QTTAAAAATFSEQVGGGSGGAGR.G K.QLIVELVVNK.M K.TYATAEPFIDAK.Y K.EMLSSTTYPVVVK.M R.VLLVIDEPHTDWAK.Y K.TNTGSAMLEQIAMSDR.Y K.VDNQHDFQDIASVVALTK.T K.QTTAAAAATFSEQVGGGSGGAGR.G K.LGTEEFPLIDQTFYPNHK.E R.ASTAAPVASPAAPSPGSSGGGGFFSSLSNAVK.Q K.TYATAEPFIDAK.Y K.EMLSSTTYPVVVK.M K.EMLSSTTYPVVVK.M R.VLLVIDEPHTDWAK.Y K.TNTGSAMLEQIAMSDR.Y K.TNTGSAMLEQIAMSDR.Y K.TNTGSAMLEQIAMSDR.Y K.VDNQHDFQDIASVVALTK.T K.LGTEEFPLIDQTFYPNHK.E K.LWVDTCSEIFGGLDICAVEALHGK.D K.QLIVELVVNK.M K.TYATAEPFIDAK.Y K.EMLSSTTYPVVVK.M R.VLLVIDEPHTDWAK.Y K.TNTGSAMLEQIAMSDR.Y K.VDNQHDFQDIASVVALTK.T K.LGTEEFPLIDQTFYPNHK.E R.ASTAAPVASPAAPSPGSSGGGGFFSSLSNAVK.Q R.SLKPDFVLIR.Q K.TYATAEPFIDAK.Y K.EMLSSTTYPVVVK.M R.VLLVIDEPHTDWAK.Y K.TNTGSAMLEQIAMSDR.Y K.VDNQHDFQDIASVVALTK.T K.QTTAAAAATFSEQVGGGSGGAGR.G K.LGTEEFPLIDQTFYPNHK.E R.QLITDLVISK.M R.SFRPDFVLIR.Q R.EMLTLPTFPVVVK.I K.VLLVVDEPHTDWAK.C K.FPLIEQTYYPNHR.E K.TNTGSAMLEQIAMSDR.Y K.ILGDYDIK.V R.QLITDLVISK.M R.SFRPDFVLIR.Q R.EMLTLPTFPVVVK.I K.VLLVVDEPHTDWAK.C K.FPLIEQTYYPNHR.E K.QTAASAGLVDAPAPSAASR.K K.TNTGSAMLEQIAMSDR.Y K.SQSLTNAFSFSESSFFR.S K.DYIFEVMDCSMPLIGEHQVEDR.Q K.VEQAEFSELNLVAHADGTYAVDMQVLR.N R.QLITDLVISK.M K.VLLVVDEPHTDWAK.C K.FPLIEQTYYPNHR.E

Oxidation: M259, M344

(1)

115

18

50

63.5

8.73

293

12

(2 + 3)

112

19

47

63.5

8.73

621

32

(4)

135

21

49

63.5

8.73

211

11

Oxidation: M258, M343, M349

Oxidation: M258, M343, M349

Oxidation: M343, M349

Oxidation: M258, M343, M349

Deamidation: N158

Oxidation: M166, M259, M344, M350

W.-F. Diao et al. / Neurochemistry International 52 (2008) 1002–1011

1007

Table 1 (Continued ) Protein name

Synapsin IIb

Accession Number

Q63537-2

spot number

MS score

Peps

MS%

MWt (KDa)

PIt

(5)

156

27

52

63.5

8.73

(6)

76

16

37

63.5

(7)

70

10

25

(8)

207

22

(1)

65

(2)

MS/MS score

MS/MS (%)

MS/MS peptide sequence

Posttranslational modifications

920

38

Oxidation: M166, M259, M344, M350, M364

8.73

665

30

63.5

8.73

627

35

51

63.5

8.73

504

22

K.ILGDYDIK.V K.KILGDYDIK.V R.QLITDLVISK.M R.SFRPDFVLIR.Q R.EMLTLPTFPVVVK.I K.VLLVVDEPHTDWAK.C K.FPLIEQTYYPNHR.E K.TNTGSAMLEQIAMSDR.Y K.QTAASAGLVDAPAPSAASR.K K.SQSLTNAFSFSESSFFR.S K.LWVDACSEMFGGLDICAVK.A K.DYIFEVMDCSMPLIGEHQVEDR.Q K.VEQAEFSELNLVAHADGTYAVDMQVLR.N K.VENHYDFQDIASVVALTQTYATAEPFIDAK.Y R.QLITDLVISK.M R.SFRPDFVLIR.Q R.EMLTLPTFPVVVK.I K.VLLVVDEPHTDWAK.C K.FPLIEQTYYPNHR.E K.TNTGSAMLEQIAMSDR.Y K.QTAASAGLVDAPAPSAASR.K K.SQSLTNAFSFSESSFFR.S K.DYIFEVMDCSMPLIGEHQVEDR.Q K.VEQAEFSELNLVAHADGTYAVDMQVLR.N R.QLITDLVISK.M R.SFRPDFVLIR.Q K.VLLVVDEPHTDWAK.C K.FPLIEQTYYPNHR.E K.TNTGSAMLEQIAMSDR.Y K.QTAASAGLVDAPAPSAASR.K K.SQSLTNAFSFSESSFFR.S K.DYIFEVMDCSMPLIGEHQVEDR.Q K.VEQAEFSELNLVAHADGTYAVDMQVLR.N R.QLITDLVISK.M R.SFRPDFVLIR.Q R.EMLTLPTFPVVVK.I K.VLLVVDEPHTDWAK.C K.FPLIEQTYYPNHR.E K.QTAASAGLVDAPAPSAASR.K K.TNTGSAMLEQIAMSDR.Y K.SQSLTNAFSFSESSFFR.S K.LWVDACSEMFGGLDICAVK.A

11

41

63.5

8.73

85

9

107

16

54

63.5

8.73

114

4

(3)

69

10

29

63.5

8.73

96

4

(4)

68

13

44

63.5

8.73

137

6

(5)

103

14

37

63.5

8.73

277

25

R.QLITDLVISK.M R.SFRPDFVLIR.Q R.EMLTLPTFPVVVK.I K.FPLIEQTYYPNHR.E K.ILGDYDIK.V R.QLITDLVISK.M R.SFRPDFVLIR.Q K.ILGDYDIK.V R.QLITDLVISK.M R.SFRPDFVLIR.Q K.ILGDYDIK.V R.QLITDLVISK.M R.SFRPDFVLIR.Q R.QHAFGMAENEDFR.H R.TSISGNWK.T K.ILGDYDIK.V R.QLITDLVISK.M R.SFRPDFVLIR.Q R.EMLTLPTFPVVVK.I R.QHAFGMAENEDFR.H K.VLLVVDEPHTDWAK.C K.FPLIEQTYYPNHR.E K.QTAASAGLVDAPAPSAASR.K K.TNTGSAMLEQIAMSDR.Y

Oxidation: M259, M344, M350

Oxidation:M344, M350

Oxidation: M259, M344, M350, M364

1008

W.-F. Diao et al. / Neurochemistry International 52 (2008) 1002–1011

Table 1 (Continued ) Protein name

Accession Number

spot number

MS score

Peps

MS%

MWt (KDa)

PIt

MS/MS score

(6)

168

21

(7)

314

(8)

MS/MS (%)

53

63.5

8.73

33

4

24

51

63.5

8.73

160

7

179

21

49

63.5

8.73

44

4

MS/MS peptide sequence R.SFRPDFVLIR.Q K.FPLIEQTYYPNHR.E R.SFRPDFVLIR.Q R.QHAFGMAENEDFR.H K.FPLIEQTYYPNHR.E R.SFRPDFVLIR.Q K.FPLIEQTYYPNHR.E

Synaptotagmin-1

P21707

69

9

27

47.4

8.57

340

24

K.LGDICFSLR.Y K.MDVGGLSDPYVK.I K.TLVMAVYDFDR.F K.VQVVVTVLDYDK.I R.RPIAQWHTLQVEEEVDAMLAVK.K

Synuclein, beta

Q5PPN9

79

2

20

14.2

4.41

399

38

K.EGVLYVGSK.T K.TKEGVLYVGSK.T K.EGVVQGVASVAEK.T K.TKEGVVQGVASVAEK.T K.EQASHLGGAVFSGAGNIAAATGLVK.K K.TKEQASHLGGAVFSGAGNIAAATGLVK.K

ERC protein 2-1

Q8K3M6-1

(1)

110.6

6.62

326

8

(2)

110.6

6.62

551

14

(3)

110.6

6.62

156

4

21.7

4.50

71

12

K.APAPAAPAAEPQAEAPVASSEQSVAVKE.-

61.2

4.63

1189

47

R.FASFIER.V R.EGLEETLR.N K.TLEIEACR.G R.ALYEQEIR.D R.YEEEVLSR.E R.FTVLTESAAK.N K.EYQDLLNVK.M K.VLEAELLVLR.Q K.MALDIEIAAYR.K K.NMQNAEEWFK.S R.IDSLMDEIAFLK.K K.RIDSLMDEIAFLK.K M.SSFSYEPYFSTSYK.R R.SAYSGLQSSSYLMSAR.A R.SAYSSYSAPVSSSLSVR.R R.LSFTSVGSITSGYSQSSQVFGR.S K.QNADISAMQDTINKLENELR.S K.QLQELEDKQNADISAMQDTINK.L R.SYSSSSGSLMPSLENLDLSQVAAISNDLK.S K.VLEAELLVLR.Q K.MALDIEIAAYR.K R.IDSLMDEIAFLK.K R.SAYSGLQSSSYLMSAR.A R.SAYSSYSAPVSSSLSVR.R

Brain acid soluble protein 1

Q05175

Neurofilament triplet L protein

P19527

(1)

(2)

64

147

9

21

20

41

61.2

4.63

411

22

Posttranslational modifications

Oxidation: M226

K.IAELESLTLR.H R.DLEDEIQMLK.A R.AAILQTEVDALR.L K.DANIALLELSASK.K R.GAEHFTIELTEENFR.R K.TQEEVMALK.R K.IAELESLTLR.H K.QEALLAAISEK.D R.DLEDEIQMLK.A R.AAILQTEVDALR.L K.DANIALLELSASK.K R.DSTMLDLQAQLK.E R.GAEHFTIELTEENFR.R R.MAEAESQVSHLEVILDQK.E K.SLQTDSSNTDTALATLEEALSEK.E R.AAILQTEVDALR.L K.DANIALLELSASK.K R.GAEHFTIELTEENFR.R

Oxidation: M65, M217, M274, M381, M435, Y178

Deamidation: Q93

Oxidation: M217, M381, M435

Accession number, protein name, MWt (theoretical molecular weight) and PIt (theoretical isoelectric point) are retrieved from UniProtKB database. MS score, Peps (matched peptides), MS% (MS coverage rate), MS/MS score, MS/MS% (MS/MS coverage rate), MS/MS peptide sequences and posttranslational modifications are obtained from MASCOT search results. Minimum MASCOT criteria for identification: MS/MS score higher than 25, MS score higher than 63.

Table 2 Quantification result of differentially expressed neuronal proteins of rat hippocampus Spot number

M (mean  S.D.)

PE (mean  S.D.)

E (mean  S.D.)

ME1 (mean  S.D.)

ME2 (mean  S.D.)

DE (mean  S.D.)

P value Of ANOVA

Synapsin-1

(1) (2) (3) (4) (5) sum

0.073  0.043 0.113  0.033 0.125  0.044 0.304  0.256 0.251  0.202 0.733  0.530

0.106  0.049 0.223  0.104 0.226  0.155 0.274  0.134 0.145  0.027 0.820  0.156

0.045  0.009 0.112  0.031 0.109  0.049 0.318  0.166 0.257  0.187 0.808  0.362

0.111  0.076 0.140  0.034 0.180  0.087 0.295  0.130 0.199  0.065 0.848  0.228

0.077  0.074 0.157  0.059 0.190  0.045 0.218  0.064 0.265  0.118 0.729  0.217

0.081  0.068 0.089  0.027 0.174  0.094 0.204  0.016 0.178  0.097 0.576  0.306

0.341 0.002 0.201 0.649 0.467 0.740

(1) (2 + 3) (4) (5) (6) (7) (8) Sum

0.200  0.097 0.263  0.102 0.191  0.054 0.214  0.077 0.250  0.091 0.258  0.101 0.283  0.134 1.438  0.636

0.166  0.040 0.178  0.054 0.205  0.054 0.244  0.105 0.221  0.108 0.465  0.201 0.442  0.214 1.869  0.594

0.197  0.063 0.157  0.058 0.152  0.034 0.213  0.032 0.175  0.047 0.286  0.120 0.272  0.083 1.403  0.373

0.265  0.213 0.228  0.069 0.194  0.084 0.212  0.077 0.183  0.058 0.260  0.119 0.285  0.132 1.574  0.477

0.205  0.111 0.246  0.092 0.166  0.067 0.241  0.049 0.316  0.062 0.201  0.054 0.185  0.055 1.456  0.414

0.198  0.105 0.244  0.094 0.209  0.074 0.279  0.119 0.241  0.068 0.341  0.120 0.251  0.125 1.629  0.238

0.727 0.090 0.432 0.589 0.022 0.007 0.036 0.611

(1) (2) (3) (4) (5) (6) (7) (8) Sum

0.099  0.042 0.262  0.093 1.005  0.106 0.819  0.110 1.162  0.131 0.818  0.217 0.307  0.053 0.237  0.096 4.109  0.536

0.123  0.018 0.313  0.085 1.056  0.317 0.903  0.248 1.354  0.307 1.043  0.224 0.459  0.098 0.464  0.153 4.790  0.728

0.110  0.016 0.308  0.025 1.030  0.112 0.861  0.124 1.309  0.293 0.891  0.207 0.433  0.123 0.320  0.082 4.721  0.706

0.090  0.013 0.271  0.046 0.983  0.116 0.779  0.117 1.270  0.099 0.971  0.158 0.477  0.109 0.369  0.097 4.993  0.198

0.095  0.031 0.269  0.075 1.129  0.152 0.950  0.114 1.475  0.281 0.841  0.233 0.522  0.119 0.380  0.079 5.216  0.506

0.097  0.013 0.260  0.036 0.988  0.129 0.826  0.105 1.503  0.103 0.931  0.273 0.505  0.178 0.473  0.238 5.128  0.693

0.185 0.444 0.608 0.255 0.038 0.330 0.024 0.013 0.039

Synaptotagmin-1

0.128  0.040

0.178  0.066

0.187  0.033

0.151  0.035

0.195  0.047

0.153  0.020

0.030

Synuclein, beta

1.971  0.377

2.292  0.398

1.877  0.270

1.764  0.341

1.887  0.160

1.904  0.318

0.048

0.089  0.023 0.114  0.029 0.095  0.011 0.267  0.046

0.121  0.041 0.146  0.058 0.112  0.017 0.382  0.064

0.079  0.017 0.141  0.054 0.119  0.026 0.298  0.040

0.119  0.040 0.135  0.052 0.130  0.019 0.351  0.048

0.101  0.014 0.113  0.018 0.111  0.028 0.313  0.055

0.096  0.022 0.158  0.058 0.124  0.039 0.340  0.036

0.067 0.323 0.177 0.001

1.734  0.604

2.679  0.613

2.263  0.215

1.662  0.210

2.229  1.030

1.944  0.600

0.030

0.887  0.199 1.052  0.370 2.005  0.285

1.055  0.327 0.905  0.188 1.679  0.668

0.862  0.264 0.565  0.202 1.357  0.454

0.789  0.282 0.689  0.207 1.379  0.446

1.193  0.115 0.827  0.183 2.054  0.188

0.926  0.279 0.725  0.120 1.651  0.360

0.059 0.003 0.009

Synapsin IIa

Synapsin IIb

ERC protein 2-1

(1) (2) (3) Sum

Brain acid soluble protein 1 Neurofilament triplet L protein

(1) (2) Sum

M vs. PE

M vs. E

M vs. ME1

M vs. ME2

M vs. DE

*

PE vs. E

PE vs. ME1

PE vs. ME2

*

*

PE vs. DE

E vs. ME2

ME1 vs. ME2

*

*

*

*

**

*

** *

*

* *

* *

* * *

***

*

*

W.-F. Diao et al. / Neurochemistry International 52 (2008) 1002–1011

Protein name

*

**

*

Proteins showing significantly different expression between groups are listed. Relative protein expression levels resulting from software-assisted quantification are given (mean  S.D., n = 10 for each group). With stringent statistical criteria, ANOVA P < 0.01 (bold and italics) were considered significant, while P > 0.01 and < 0.05 (italics) were considered trend. Blank means no significant difference between groups. *means P < 0.05, **means P < 0.01, ***means P < 0.001.

1009

1010

W.-F. Diao et al. / Neurochemistry International 52 (2008) 1002–1011

modification during analysis. Degradation during processing is highly unlikely due to the conditions applied. Methionine oxidation respected in the MS identification may not be assigned any biological significance but may be considered the result of sample processing under air. 3. Discussion The major outcome of this study is the unambiguous identification of a Synapsin-1, a Synapsin IIa, an ERC-2 and a Neurofilament triplet L protein expression form as ECdependent hippocampal proteins. A series of other proteins showed a trend (P < 0.05) but we applied stringent statistical criteria (P < 0.01) and subsequently only three proteins remained reliably EC associated or sexually regulated. Two synapsin splice variants or isoforms, were presenting with EC-dependent regulation. Synapsins are neuronal synaptic-vesicle phosphoproteins that coat synaptic vesicles, bind to the cytoskeleton, and are proposed to function in the regulation of neurotransmitter release (Sudhof et al., 1989). As shown in the results section Synapsin-1 and Synapsin IIa levels were aberrant in specific phases of the EC only. The Synapsin IIb expression forms showed trends but did not reach the level of significance set. It is intriguing to realise that individual synapsin isoforms are under dependent on the individual phases of the EC. It may be mentioned that levels of synapsins observed in this work were not sex-dependent and no sexual dimorphism was observed for most proteins of the neuronal network (Table 2 and supplementary Table 2). The several individual expression forms for one protein may represent different posttranslational modifications (ptms, John et al., 2007). ERC protein 2 (CAZ-associated structural protein 1, CAST) was represented by several spots herein and the sum of ERC protein levels was different between males and female proestrous identifying a sex-dependent protein. It is thought to be involved in the organization of the cytomatrix at the nerve terminals active zone (CAZ), a specialized site where Ca2+dependent exocytosis of neurotransmitters occurs. ERC protein has been established to regulate neurotransmitter release by binding other presynaptic active zone (AZ) proteins (Higa et al., 2007; Ohtsuka et al., 2002; Takao-Rikitsu et al., 2004). The three expression forms revealed on 2DE reflect ERC protein-2 isoform 1 and different mobility on 2DE may be due to ptms, although the sequences obtained did not provide any evidence for ptms, this may be, however, technical in nature. Neurofilament triplet L protein (NF-L) is a subunit of neurofilaments, which contributes to axonal structure and define axonal diameter. NF-L is involved in maintaining structure of dendrites and axons in neurons. Increased oxidation or reduced expression of these proteins may account for reduction of dendritic spines in the hippocampal pyramidal neurons. The difference between estrous phase and male sex indicates sexual dimorphism for this structure. Only one expression form out of the three observed was significantly EC dependent. The three individual spots for NF-L may be due to ptms and indeed, NF-L-serine acetylation has been reported by

direct submission to UniProtKB database (http://www.expasy.org/uniprot/P19527#ref4) and in the protein corresponding to NF-l spot 1 deamidation was observed. The biological meaning of this differential NF-L protein levels remains open and it remains to be shown if dendritic structure and dynamics is affected by this biochemical difference. Taken together, sex- and EC- dependent levels of three key proteins of the neuronal network were observed and this is relevant for the design of future studies in the hippocampus at the protein level respecting not only gender differences but also the estrous phase. Based upon others’ and our work, it is not sufficient to define the sex but to respect the EC. In addition, previous work on protein expression in hippocampus will have to be interpreted taking into acccount the EC differences given herein. Acknowledgment We are highly indebted to the Verein zur Durchfu¨hrung der wissenschaftlichen Forschung auf dem Gebiet der Neonatologie und Kinderintensivmedizin ‘‘Unser Kind’’. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neuint.2007. 10.013. References Adams, M.M., Morrison, J.H., Gore, A.C., 2001. N-methyl-D-aspartate receptor mRNA levels change during reproductive senescence in the hippocampus of female rats. Exp. Neurol. 170, 171–179. Baker, D.E.J., 1979. Biology and diseases. In: Baker, H.J., Lindsey, J.R., Weisbroth, S.H. (Eds.), The Laboratory Rat. Academic Press, New York, pp. 153–168. Berry, B., McMahan, R., Gallagher, M., 1997. Spatial learning and memory at defined points of the estrous cycle: effects on performance of a hippocampal-dependent task. Behav. Neurosci. 111, 267–274. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Chen, W.Q., Kang, S.U., Lubec, G., 2006. Protein profiling by the combination of two independent mass spectrometry techniques. Nat. Protocols 1, 1447– 1452. Chesler, E.J., Juraska, J.M., 2000. Acute administration of estrogen and progesterone impairs the acquisition of the spatial morris water maze in ovariectomized rats. Horm. Behav. 38, 234–242. Cooke, B.M., Woolley, C.S., 2005. Gonadal hormone modulation of dendrites in the mammalian CNS. J. Neurobiol. 64, 34–46. Crispino, M., Stone, D.J., Wei, M., Anderson, C.P., Tocco, G., Finch, C.E., Baudry, M., 1999. Variations of synaptotagmin I, synaptotagmin IV, and synaptophysin mRNA levels in rat hippocampus during the estrous cycle. Exp. Neurol. 159, 574–583. Devidze, N., Mong, J.A., Jasnow, A.M., Kow, L.M., Pfaff, D.W., 2005. Sex and estrogenic effects on coexpression of mRNAs in single ventromedial hypothalamic neurons. Proc. Natl. Acad. Sci. USA 102, 14446–14451. Frischer, T., Myung, J.K., Maurer, G., Eichler, I., Szepfalusi, Z., Lubec, G., 2006. Possible dysregulation of chaperon and metabolic proteins in cystic fibrosis bronchial tissue. Proteomics 6, 3381–3388.

W.-F. Diao et al. / Neurochemistry International 52 (2008) 1002–1011 Gulesserian, T., Engidawork, E., Fountoulakis, M., Lubec, G., 2007. Manifold decrease of sialic acid synthase in fetal Down syndrome brain. Amino Acids 32, 141–144. Halpern, D.F., Tan, U., 2001. Stereotypes and steroids: using a psychobiosocial model to understand cognitive sex differences. Brain Cogn. 45, 392–414. Higa, S., Tokoro, T., Inoue, E., Kitajima, I., Ohtsuka, T., 2007. The active zone protein CAST directly associates with ligand-of-numb protein X. Biochem. Biophys. Res. Commun. 354, 686–692. Jahn, R., 2004. Principles of exocytosis and membrane fusion. Ann. N.Y. Acad. Sci. 1014, 170–178. John, J.P., Chen, W.Q., Pollak, A., Lubec, G., 2007. Mass spectrometric studies on mouse hippocampal synapsins Ia, IIa, and IIb and identification of a novel phosphorylation site at serine-546. J. Proteome. Res. 6, 2695–2710. Korol, D.L., Kolo, L.L., 2002. Estrogen-induced changes in place and response learning in young adult female rats. Behav. Neurosci. 116, 411–420. Maeda, K.-I., Ohkura, S., Tsukamura, H., 2000. Physiology of reproduction. In: Krinke, G.J. (Ed.), The Laboratory Rat: The Handbook of Experimental Animals. Academic Press, London, pp. 145–176. Mirnics, K., Middleton, F.A., Lewis, D.A., Levitt, P., 2001. Analysis of complex brain disorders with gene expression microarrays: schizophrenia as a disease of the synapse. Trends Neurosci. 24, 479–486. Mirnics, K., Middleton, F.A., Marquez, A., Lewis, D.A., Levitt, P., 2000. Molecular characterization of schizophrenia viewed by microarray analysis of gene expression in prefrontal cortex. Neuron 28, 53–67. Mizuno, K., Giese, K.P., 2005. Hippocampus-dependent memory formation: do memory type-specific mechanisms exist? J. Pharmacol. Sci. 98, 191–197. Myung, J.K., Lubec, G., 2006. Use of solution-IEF-fractionation leads to separation of 2673 mouse brain proteins including 255 hydrophobic structures. J. Proteome. Res. 5, 1267–1275. Ohtsuka, T., Takao-Rikitsu, E., Inoue, E., Inoue, M., Takeuchi, M., Matsubara, K., Deguchi-Tawarada, M., Satoh, K., Morimoto, K., Nakanishi, H., Takai, Y., 2002. Cast: a novel protein of the cytomatrix at the active zone of synapses that forms a ternary complex with RIM1 and munc13-1. J. Cell Biol. 158, 577–590.

1011

Royle, S.J., Lagnado, L., 2003. Endocytosis at the synaptic terminal. J. Physiol. 553, 345–355. Sandstrom, N.J., Williams, C.L., 2001. Memory retention is modulated by acute estradiol and progesterone replacement. Behav. Neurosci. 115, 384–393. Stackman, R.W., Blasberg, M.E., Langan, C.J., Clark, A.S., 1997. Stability of spatial working memory across the estrous cycle of Long-Evans rats. Neurobiol. Learn. Mem. 67, 167–171. Sudhof, T.C., 2000. The synaptic vesicle cycle revisited. Neuron 28, 317–320. Sudhof, T.C., 2004. The synaptic vesicle cycle. Annu. Rev. Neurosci. 27, 509– 547. Sudhof, T.C., Czernik, A.J., Kao, H.T., Takei, K., Johnston, P.A., Horiuchi, A., Kanazir, S.D., Wagner, M.A., Perin, M.S., De Camilli, P., et al., 1989. Synapsins: mosaics of shared and individual domains in a family of synaptic vesicle phosphoproteins. Science 245, 1474–1480. Takao-Rikitsu, E., Mochida, S., Inoue, E., Deguchi-Tawarada, M., Inoue, M., Ohtsuka, T., Takai, Y., 2004. Physical and functional interaction of the active zone proteins, CAST, RIM1, and Bassoon, in neurotransmitter release. J. Cell Biol. 164, 301–311. Virmani, T., Gupta, P., Liu, X., Kavalali, E.T., Hofmann, S.L., 2005. Progressively reduced synaptic vesicle pool size in cultured neurons derived from neuronal ceroid lipofuscinosis-1 knockout mice. Neurobiol. Dis. 20, 314– 323. Warren, S.G., Juraska, J.M., 1997. Spatial and nonspatial learning across the rat estrous cycle. Behav. Neurosci. 111, 259–266. Wood, G.E., Beylin, A.V., Shors, T.J., 2001. The contribution of adrenal and reproductive hormones to the opposing effects of stress on trace conditioning in males versus females. Behav. Neurosci. 115, 175–187. Woolley, C.S., 1998. Estrogen-mediated structural and functional synaptic plasticity in the female rat hippocampus. Horm. Behav. 34, 140–148. Woolley, C.S., McEwen, B.S., 1992. Estradiol mediates fluctuation in hippocampal synapse density during the estrous cycle in the adult rat. J. Neurosci. 12, 2549–2554. Yang, J.W., Czech, T., Lubec, G., 2004. Proteomic profiling of human hippocampus. Electrophoresis 25, 1169–1174.