Dysregulated CREB signaling pathway in the brain of neural cell adhesion molecule (NCAM)-deficient mice

Dysregulated CREB signaling pathway in the brain of neural cell adhesion molecule (NCAM)-deficient mice

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Research Report

Dysregulated CREB signaling pathway in the brain of neural cell adhesion molecule (NCAM)-deficient mice Anu Aonurm-Helm, Tamara Zharkovsky, Monika Jürgenson, Anti Kalda, Alexander Zharkovsky⁎ Department of Pharmacology, Centre of Excellence for Translational Medicine, University of Tartu, 19 Ravila Street, 51014, Estonia

A R T I C LE I N FO

AB S T R A C T

Article history:

The neural cell adhesion molecule (NCAM) mediates cell–cell interactions and plays an

Accepted 30 August 2008

important role in processes associated with neural plasticity, including learning and

Available online 12 September 2008

memory formation. It has been shown that mice deficient in all isoforms of NCAM (NCAM−/− mice) demonstrate impairment in long-term plasticity at multiple hippocampal synapses,

Keywords:

disrupted spatial learning, and impaired contextual and auditory-cued fear conditioning.

Neural cell adhesion

The formation of long-term memory is associated with activation of transcription factor

molecule (NCAM)

CREB (cAMP response element binding protein). The aims of this study were to investigate

NCAM-deficient mice

NCAM-mediated signaling transduction pathways and the levels of the phosphorylated

pCREB

(Ser133) active form of the CREB in the brain structures (the pre- and frontal cortex,

CaMKII

basolateral amygdala, and hippocampus) involved in the memory formation in NCAM-

CaMKIV

deficient mice. Immunohistochemical analysis revealed reduced levels of pCREB in the prefrontal cortex (PFC), frontal cortex (FC), CA3 subregion of the hippocampus (CA3) and basolateral nucleus of amygdala (BLA) in NCAM−/− mice. NCAM−/− mice had also reduced levels of the phosphorylated CaMKII and CaMKIV in PFC/FC and the hippocampus, which are the downstream signaling molecules of NCAM. The levels of non-phosphorylated kinases did not differ from those seen in the wild-type mice. These results provide evidence that NCAM deficiency results in the dysregulation of CREB-mediated signaling pathways in the brain regions, which is related to the formation of memory. © 2008 Elsevier B.V. All rights reserved.

1.

Introduction

The neural cell adhesion molecule (NCAM) is a membraneassociated glycoprotein expressed on the surface of neurons and glial cells. The NCAM is a member of the immunoglobulin super-family of adhesion molecules (Edelman, 1986) and plays a major role in cell-to-cell and cell to extra-cellular matrix interactions (Crossin and Krushel, 2000). Alternative splicing of a single gene yields three main NCAM isoforms: 180- and 140 kDa transmembrane isoforms and a 120 kDa glucophosphatidyl inositol-linked isoform (Walmod et al., 2004).

Adhesive properties of NCAM can be regulated through the addition of long linear homopolymers of alfa-2,8-linked sialic acid residues (polysialic acid or PSA) (Rutishauser, 1996), which attenuates NCAM-mediated cell interaction and thereby creates plasticity in the positioning and movements of the cells and/or their processes (Rutishauser, 1996; Bruses and Rutishauser, 2001). NCAM is widely expressed in the central nervous system in which it mediates several neuronal functions by controlling intercellular adhesion, neurite outgrowth, and cell migration, proliferation and survival triggered by homophilic interactions as well as by the heterophilic

⁎ Corresponding author. Fax: +3727 374352. E-mail address: [email protected] (A. Zharkovsky). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.08.091

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Fig. 1 – Representative microphotograph demonstrating pCREB-positive cells in the prefrontal cortex (PFC), frontal cortex (FC), CA3 subregion of hippocampus (CA3) and basolateral amygdala (BLA) of NCAM+/+ and NCAM−/− mice.

binding of NCAM to other adhesion molecules, extra-cellular matrix components and cell surface receptors (Cambon et al., 2004; Walmod et al., 2004; Ditlevsen et al., 2008). NCAM promotes neuronal plasticity via interaction and activation of the FGF receptor, focal adhesion kinase (FAK), growth cone associated protein (GAP-43) and Src family of tyrosine kinase, Fyn (Doherty and Walsh, 1996; Viollet and Doherty, 1997; Beggs et al., 1994, He and Meiri, 2002; Walmod et al., 2004). Homophilic and heterophilic interactions of NCAM initiate signaling transduction pathways like mitogen-activated protein kinase (MAPK) cascade and calcium/calmodulin dependent kinase II (CaMKII) and calcium/calmodulin dependent kinase IV (CaMKIV) (Schmid et al., 1999; Kolkova et al., 2000; Povlsen et al., 2003; Griffith, 2004; Ditlevsen et al., 2008). Several lines of evidence have also demonstrated a crucial role of NCAM in the formation of memory. The administration of antibodies directed against NCAM or PSA-NCAM into the hippocampus induced amnesia in rats and interfered with the consolidation of memory in rats (Doyle et al., 1992). Mice deficient in all isoforms of NCAM have demonstrated impairment in long-term plasticity at the mossy fiber synapses and disrupted learning abilities in the Morris water maze (Lüthl et

al., 1994; Cremer et al., 1994, 1998; Stork et al., 2000) and impaired contextual and auditory-cued fear conditioning (Stork et al., 2000; Welzl and Stork, 2003; Senkov et al., 2006). Spatial memory impairment and reduction in LTP were also observed in rats with conditional ablation of NCAM (Bukalo et al., 2004). Stimulation of NCAM homophilic and heterophilic binding to the FGF receptor by synthetic peptides derived from the NCAM molecule in rodents facilitates memory consolidation, enhances social memory retention and reduces neuropathological signs and cognitive impairment induced by βamyloid25–35 (Cambon et al., 2004; Secher et al., 2006; Klementiev et al., 2007; for rev. see also Berezin and Bock, 2008). Furthermore, upregulation of NCAM polysialylation was observed following auditory and contextual fear conditioning in the amygdala and dorsal hippocampus (Markram et al., 2007; Lopez-Fernandez et al., 2007). A transient upregulation of NCAM and PSA-NCAM levels were found in the rat hippocampus following spatial training in the Morris water maze (O'Connell et al., 1997; Venero et al., 2006). Previous studies demonstrated that activation of the NCAM-mediated signaling pathways results in the increased phosphorylation of CREB (cAMP response element binding protein) at Ser133 and increased expression of c-fos (Schmid et al., 1999; Jessen et al., 2001). Transcription factor CREB, which modulates the transcription of genes with cAMP response elements in their promoters, mediates critical components of neuronal survival and memory consolidation in mammals. CREB-dependent transcription is required for cellular events underlying a variety of forms of memory, including spatial and social learning, thus indicating that CREB may be a universal modulator of processes required for memory formation (Frank and Greenberg, 1994; Silva et al., 1998; Kandel, 2001; Hall et al., 2001; Mizuno et al., 2002; Florian et al., 2008; Bernabeu et al., 1997; Impey et al., 1998). It is not known yet whether the poor ability to learn of NCAM-deficient mice is related to the persistent alterations in CREB activity and/or transduction signaling pathways linking NCAM with CREB. This led us to explore in detail the NCAMmediated signaling transduction pathways and the levels of the phosphorylated (Ser133) active form of CREB in the brain

Table 1 – Number of pCREB-positive cells in the brain regions of NCAM+/+ and NCAM−/− mice Brain region

NCAM+/+

NCAM−/−

PFC FC DG CA1 CA3 BLA BMA Pir

45.4 ± 2.6 45.4 ± 3.3 102.2 ± 4.4 49.5 ± 18.6 193.6 ± 21.2 59.1 ± 1.1 48.7 ± 1.9 70.2 ± 4.2

20.2 ± 0.7⁎⁎⁎ 18.4 ± 0.4⁎⁎ 100.7 ± 4.6 31.2 ± 4.9 25.0 ± 2.2⁎⁎⁎ 21.6 ± 1.0⁎⁎⁎ 46.9 ± 3.3 65.7 ± 3.1

The data are expressed as mean ± SEM per 0.1 mm2. ⁎⁎p < 0.01; ⁎⁎⁎p < 0.001 (Student's t-test; n = 5). Abbreviations: PFC — prefrontal cortex; FC — frontal cortex; DG — dentate gyrus of hippocampus; CA1 — CA1 subregion of hippocampus; CA3 — CA3 subregion of hippocampus; BLA — basolateral nucleus of amygdala; BMA — basomedial nucleus of amygdala; Pir — piriform cortex.

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type littermates. Cell counts for pCREB-positive cells in the frontal cortex (FC), prefrontal cortex (PFC), basolateral nucleus of the amygdala (BLA) and CA3 subregion of the hippocampus (CA3) were significantly lower in NCAM−/− mice as compared to their wild-type littermates (Student's t-test PFC p < 0.001; FC p < 0.001; BLA p < 0.0001; CA3 p < 0.0001) (Fig. 1, Table 1). In contrast, pCREB-positive cell counts in the basomedial nucleus of the amygdala (BMA), dentate gyrus of the hippocampus (DG), CA1 subregion of the hippocampus (CA1) and piriform cortex (Pir) did not differ in NCAM+/+ and NCAM−/− mice (Table 1). Since the observed reduction of pCREB expression in NCAM−/− mice might be due either to the decreased phosphorylation of CREB or to the reduced expression of CREB, we performed Western Blot analysis with lysates obtained from pooled PFC/ FC and the hippocampus of NCAM+/+ and NCAM−/− mice. Fig. 2 shows representative immunoblots of pCREB and CREB. No differences in the immunoreactivity of CREB were observed in NCAM+/+ and NCAM−/− mice (Student's t-test, p N 0.05), whereas immunoreactivity of pCREB was significantly lower in PFC/FC and the hippocampus of NCAM−/− mice (Student's ttest, PFC and FC p < 0.001; hippocampus p < 0.0001) (Fig 2).

2.2. Cell densities in different brain regions of NCAM+/+ and NCAM−/− mice To determine whereas the reduction in CREB phosphorylation in different brain regions of NCAM−/− mice is due to the reduced cell density, we performed cell density counting in NCAM−/− mice and their wild-type littermates. No differences were observed in cell densities between NCAM−/− mice and their wild-type littermates in any brain region under examination (Table 2). It seems, therefore, that the decreased number of pCREB-positive cells is not due to reduced total cell density. Fig. 2 – Western Blot analysis highlighting phosphorylation changes of CREB with total unphosphorylated protein in prefrontal/frontal cortex and hippocampus of NCAM+/+ and NCAM−/− mice (A). pCREB/CREB immunoreactivity ratio as a percent of control in prefrontal/frontal cortex (B) and hippocampus (C) of NCAM+/+ and NCAM−/− mice. CREB immunoreactivity as a % of control in prefrontal/frontal cortex (D) and hippocampus (E) of NCAM+/+ and NCAM−/− mice. *p < 0.05 (Student's t-test; n = 4).

structures (the pre- and frontal cortex, basolateral amygdala, and hippocampus) of NCAM−/− mice.

2.

Results

2.1.

pCREB expression in NCAM−/− and NCAM+/+ mice

The levels of CREB phosphorylation were estimated by two methods, which were quantitative immunohistochemistry, using phosphospecific antibodies raised against pCREB at Ser133, and semiquantitative Western immunoblotting of pCREB and CREB. We counted the pCREB immunoreactive cells in several brain regions of NCAM−/− mice and their wild-

2.3. NCAM-mediated signaling pathways in NCAM+/+ and NCAM−/− mice To understand, which signaling pathways are involved in the reduced phosphorylation of CREB in NCAM−/− mice, we performed Western Blot analyses from tissue lysates obtained from pooled PFC/FC and the hippocampus of NCAM+/+ and NCAM−/− mice for MAPK and calciumcalmodulin-dependent protein kinase (CaMK) signaling

Table 2 – Cell density (cells/mm3) in different brain regions of NCAM+/+ and NCAM−/− mice Cell density (cells/mm3) PFC FC DG CA1 CA2 CA3 BLA

NCAM+/+

NCAM−/−

280,500 ± 9877 253,500 ± 16,260 1,364,000 ± 98,500 1,433,000 ± 110,900 1,453,000 ± 120,000 1,243,000 ± 99,430 495,000 ± 1177

273,700 ± 5370 244,900 ± 14,340 1,179,000 ± 98,733 1,362,000 ± 83,420 1,295,000 ± 143,800 1,206,000 ± 57,080 477,100 ± 9346

The data are expressed as mean ± SEM (Student's t-test; n = 5). Abbreviations: PFC — prefrontal cortex; FC — frontal cortex; DG — dentate gyrus of hippocampus; CA1 — CA1 subregion of hippocampus; CA3 — CA3 subregion of hippocampus; BLA — basolateral nucleus of amygdala.

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Fig. 4 – Western Blot analysis highlighting phosphorylation of ERK with total unphosphorylated protein in prefrontal/frontal cortex and hippocampus of NCAM+/+ and NCAM−/− mice (A). pERK/ERK ratio as % of control in prefrontal/frontal cortex (B) and hippocampus (C) of NCAM+/+ and NCAM−/− mice, ERK immunoreactivity as a % of control in prefrontal/frontal cortex (D) and hippocampus (E) of NCAM+/+ and NCAM−/− mice. Student's t-test; n = 4.

Fig. 3 – Western Blot analysis highlighting phosphorylation of MEK1/2 with total unphosphorylated protein in prefrontal/ frontal cortex and hippocampus of NCAM+/+ and NCAM−/− mice (A), pMEK1/MEK ratio as a % of control in prefrontal/frontal cortex (B) and hippocampus (C) of NCAM+/+ and NCAM−/− mice, pMEK2/MEK ratio as a % of control in prefrontal/frontal cortex (D) and hippocampus (E) of NCAM+/+ and NCAM−/− mice, MEK immunoreactivity as a % of control in prefrontal/frontal cortex (F) and hippocampus (G) of NCAM +/+ and NCAM−/− mice. Student's t-test; n = 4.

pathways, which are dependent on NCAM signaling (For reviews see Ditlevsen et al., 2008). No differences were observed in the levels of pMEK1, pMEK2 and MEK, pERK and ERK, pAkt and Akt immunoreactivities (Figs. 3, 4, and 5). By contrast, a significant decrease was observed in pCaMKII and pCaMKIV immunoreactivities (Figs. 6 and 7) in NCAM−/− mice compared to their wild-type littermates, whereas no differences were observed in nonphosphorylated CaMKII and CaMKIV between NCAM+/+ and NCAM−/− mice.

3.

Discussion

Previous studies have shown that NCAM is required for the establishment of durable memories (Roullet et al., 1997) and

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NCAM-mediated CREB phosphorylation occurs under in vivo conditions, and NCAM deficiency leads to the constitutively reduced levels pCREB in brain regions playing important roles in spatial and aversive learning. We therefore propose that the observed reduction in CREB phosphorylation seen in NCAM−/− mice might be important for the impaired learning in these animals. Previous studies have demonstrated that CREB activation (phosphorylation) is mediated by two major pathways: the cAMP signaling pathway and calcium-calmodulindependent protein kinase pathway (Gonzalez and Montminy, 1989; Soderling, 2000) and that both signaling pathways are important for memory formation (Malenka et al., 1989). Previous studies demonstrated that CaMK IV knockout mice had reduced fear memory whereas pain sensitivity remained

Fig. 5 – Western Blot analysis highlighting phosphorylation of Akt with total unphosphorylated protein in prefrontal/frontal cortex and hippocampus of NCAM+/+ and NCAM−/− mice (A). pAkt/Akt ratio as a % of control in prefrontal/frontal cortex (B) and hippocampus (C) of NCAM+/+ and NCAM−/− mice, Akt immunoreactivity as a % of control in prefrontal/frontal cortex (D) and hippocampus (E) of NCAM+/+ and NCAM−/− mice. Student's t-test; n = 4.

NCAM−/− mice show reduced synaptic plasticity, which leads to memory deficits and impaired learning ability (Cremer et al., 1994, 1998; Lüthl et al., 1994; Stork et al., 2000; Welzl and Stork, 2003; Senkov et al., 2006). The present study demonstrates that NCAM−/− mice have reduced basal levels of phosphorylated transcription factor CREB. The reduced number of cells expressing pCREB was region-specific and found in regions, which are associated with memory formation: PFC, FC, CA3 and BLA (Hotte et al., 2006). By contrast, DG, CA1, BMA or Pir of NCAM−/− and NCAM+/+ mice had a similar basal number of pCREB-positive cells. Previous studies using neuroblastoma cell lines or primary hippocampal neurons have demonstrated the activation of NCAM induces CREB phosphorylation at serine 133 (Schmid et al., 1999; Jessen et al., 2001). Our data thus demonstrate that

Fig. 6 – Western Blot analysis highlighting phosphorylation changes of CaMKII with total unphosphorylated protein in prefrontal/frontal cortex and hippocampus of NCAM+/+ and NCAM−/− mice (A). pCaMKII/CaMKII ratio as a % of control in prefrontal/frontal cortex (B) and hippocampus (C) of NCAM+/+ and NCAM−/− mice, CaMKII immunoreactivity as a % of control in prefrontal/frontal cortex (D) and hippocampus (E) of NCAM+/+ and NCAM−/− mice. *p < 0.05 (Student's t-test; n = 4).

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levels of CREB phosphorylation and impaired ability of NCAM knockout mice to learn. In contrast, we failed to demonstrate any alterations in pMEK1, pMEK2, MEK, pERK and ERK, pAkt and Akt immunoreactivities in the brain of NCAM−/− mice. It seems that the basal activity of the MAP kinase pathway is not largely affected by NCAM deficiency. Since in the present study, only basal levels of activity of CREB and transduction signaling pathways were studied, we don't know yet how signaling pathways will be affected by memory acquisition or retrieval in NCAM-deficient mice. These studies are now being conducted in our laboratory. In conclusion, our data demonstrate that NCAM deficiency results in the reduced levels of the phosphorylated transcription factor CREB and reduced levels of phosphorylated CaMKII and CaMKIV in the brain regions involved in the formation and maintenance of memories. We propose that the reduction in pCREB levels is due to the impairment in the calciumcalmodulin-dependent protein kinase pathway and this might account for the impaired learning ability in NCAMdeficient mice.

Fig. 7 – Western Blot analysis highlighting phosphorylation changes of CaMKIV with total unphosphorylated protein in prefrontal/frontal cortex and hippocampus of NCAM+/+ and NCAM−/− mice (A). pCaMKIV/CaMKIV ratio as a % of control in prefrontal/frontal cortex (B) and hippocampus (C) of NCAM+/+ and NCAM−/− mice, CaMKIV immunoreactivity as a % of control in prefrontal/frontal cortex (D) and hippocampus (E) of NCAM+/+ and NCAM−/− mice. *p < 0.05 (Student's t-test; n = 4).

unchanged (Wei et al., 2002). It has been also shown that mice deficient in CaMK kinase alpha exhibit a deficit in Pavlovian fear conditioning and that these animals show impaired activation of CaMKIV (Blaeser et al., 2006). Defects in LTP and impairments in spatial learning occur in mice deficient in CaMKII (Silva et al., 1992). Furthermore previous studies have demonstrated that NCAM- or FGF-mediated increase in [Ca2+]i results in the activation of CaMKII since NCAM-mediated neuritogenesis can be blocked by the Ca2+/calmodulin-dependent protein kinase inhibitor KN-62 (Williams et al., 1995). Our study showed that NCAM-deficient mice have decreased levels of phosphorylated or an active form of CaMKII and CaMKIV proteins and this might account for the observed decreased

4.

Experimental procedures

4.1.

Subjects

All experimental procedures were approved by the Ethical Committee of University of Tartu (Tartu, Estonia) and were carried out by individuals who hold an appropriate license. NCAM−/− mice and NCAM+/+ mice used for this study were obtained by crossing C57BL/6-Ncamtm1Cgn+/− heterozygotic mice purchased from Jackson Laboratories, Main, US). F2 generation NCAM−/− mice and their wild-type (NCAM+/+) littermates at the age 4–6 months and with an average weight of 22.0 g were used. All animals were housed under standard housing conditions: mice were group-housed (5 mice per cage) under a 12-h light/dark cycle. All mice had free access to food and water.

4.2.

pCREB immunohistochemistry and cell quantification

The mice were deeply anesthetized with chloral hydrate (300 mg/kg, i.p.) and transcardially perfused using 0.9% saline and then with 4% paraformaldehyde in phosphate buffered saline (PBS, 0.1 M, pH = 7.4). After a post-fixation of the brain in paraformaldehyde/PBS solution for 24 h, sections 40 μm thick were cut on a vibromicrotome (Leica VT1000S, Germany), collected in PBS and kept at 4 °C until further processing. For pCREB immunohistochemistry, the free-floating sections were incubated in 0.3% H2O2 in PBS for 30 min and followed by unmasking with 0.01 M citrate buffer (pH = 6.0) in a water bath at 84 °C for 30 min. Incubation in blocking solution for 1 h was followed by 24-h incubation at room temperature with goat polyclonal antibody to pCREB (1:200; Santa Cruz Biotechnology Inc., Germany) diluted in blocking solution. After being washed in PBS, sections were incubated in biotinylated rabbit anti-goat antibody (1:200; Vector Laboratories, UK) diluted in blocking solution for 1 h. pCREB-positive cells were visualized using the peroxydase method (ABC system and diaminobenzidine as chromogen, Vector

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Laboratories, UK). The sections were dried, cleared with xylol and cover-slipped with mounting medium (Vector Laboratories, UK). The number of pCREB-positive nuclei were counted in the following brain areas according to Paxinos and Franklin (2001) according to bregma: the prefrontal cortex (PFC) and frontal cortex (FC), from 2.96 mm to 2.58 mm; the basolateral nucleus of the amygdala (BLA) and basomedial nucleus of the amygdala (BMA), from −1.06 mm to − 1.58 mm; hippocampus and piriform cortex (Pir), from −1.82 mm to −2.46 mm. For each structure, four random sections per animal were taken and positive nuclei were counted manually according to the optical fractionation method (West, 1993) where the number of counting frames in the delineated region were applied randomly by CAST program (Olympus, Denmark). Counting was performed using an Olympus BX-51 microscope. Immunoreactivity was expressed as the number of positive nuclei per 0.1 mm2 of brain region. At all stages of assessment, the experimenter was blind to the experimental groupings.

4.3.

Cell staining and cell density analysis

For quantification of the total cell density in different brain regions, every sixth section throughout the region was incubated in a 0.1 M Tris–HCl buffer, containing 0.025% trypsin and 0.1% CaCl2, for 10 min, followed by incubation in acid-alcohol (HCl 1% in 70% ethanol) solution for 10 s. The slides were stained using haematoxylin–eosine, washed in PBS and cover-slipped with a water-based mounting medium (Vector Laboratories, UK). Cell numbers were quantified according to the optical fractionation method (West, 1993). The stereology system consisted of an Olympus BX-51 microscope, a microcator (Heidenhain, DN 281) and the computer Assisted Stereological Toolbox (CAST-2)-Grid system (Olympus, Denmark). Numerical density (Nv) was calculated according to the formula Nv = ΣQ / Σv (dis), where ΣQ is the number of cells counted and Σv (dis) is the volume of dissectors.

4.4.

Immunoblotting analysis

Adult (4 months old) NCAM+/+ and NCAM−/− mice (both groups consisted of 4 animals) were sacrificed by decapitation for immunoblotting analysis. The brain was removed from the scull on ice and in a cold room (+4 °C). Olfactory bulbs and cerebellum were removed, and PFC and FC were dissected out approximately 1 mm from rostral part of the hemispheres according to the following coordinates: bregma from 3.56 mm to 2.58 mm (Paxinos and Franklin, 2001). PFC and FC were pooled for each probe. Hippocampi were dissected as described in the published protocol (Madison and Edson, 1997). The brain was bisected with a scalpel along the midline. The hemibrain was turned so that the medial surface was facing up, and the neocortex was peeled off, exposing the hippocampus. When the hippocampus was totally exposed, it was taken out. The dissected brain tissues were placed immediately into liquid nitrogen and stored at −80 °C until further processing. Tissues were lysed in 10 vol RIP-A lysis buffer: 20 mM Tris– HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 1% NP-40, 2 mM EDTA

containing protease and phosphatase inhibitors, homogenized manually, incubated for 20 min on ice and centrifuged (13,000 rpm for 20 min at 4 °C). The supernatants were resolved by electrophoresis on 10% or 12% SDS-polyacrylamide gel. Proteins were transferred onto Hybond™-P PVDF Transfer Membranes (Amersham Biosciences, UK) in 0.1 M Tris-base, 0.192 M glycine and 20% (w/w) methanol using an electrophoretic transfer system. The membranes were blocked with 0.1% (w/w) Tween-20/TBS containing 5% (w/w) non-fat dried milk powder at room temperature for 1 h. After blocking, the membranes were incubated overnight at 4 °C with one of the following polyclonal antibodies: goat anti pCREB (1:4000; Santa Cruz Biotechnology Inc., Germany), rabbit anti-CREB (1:2000; Santa Cruz Biotechnology Inc., Germany), goat anti p-MEK1/2 (1:800; Santa Cruz Biotechnology Inc., Germany), rabbit anti-MEK (1:800; Santa Cruz Biotechnology Inc., Germany), goat anti-pERK (1:800; Santa Cruz Biotechnology Inc., Germany), rabbit anti-ERK (1:800; Santa Cruz Biotechnology Inc., Germany), goat anti-pCaMKII (1:800; Santa Cruz Biotechnology Inc., Germany), rabbit antiCaMKII (1:800; Santa Cruz Biotechnology Inc., Germany), rabbit anti-pCaMKIV (1:800; Santa Cruz Biotechnology Inc., Germany), goat anti-CaMKIV (1:800; Santa Cruz Biotechnology Inc., Germany), rabbit anti-pAkt (1:800; Santa Cruz Biotechnology Inc., Germany) and rabbit anti-Akt (1:800; Santa Cruz Biotechnology Inc., Germany) followed by incubation with secondary antibodies: anti-goat IgG (1:10,000; Vector Laboratories, UK) and anti-rabbit-HRP (1:2000; Pierce, US), respectively for 1 h at room temperature, followed by incubation with ABC system (Vector Laboratories, UK). The membranes were incubated with ECL detection reagent (ECL, Amersham, UK) for 5 min to visualize proteins, and then exposed to autoradiography X-ray film (Amersham hyperfilm ECL, UK). To normalize the proteins' immunoreactivities, the β-actin protein was measured on the same blot with a mouse monoclonal anti-β-actin antibody (1:10,000; Sigma, St.Louis, US) followed by anti-mouse HRP secondary antibody (1:2000; Pierce, US) for 1 h at room temperature as an internal control for loading. The blots probed for proteins of interest were densitometrically analyzed using QuantityOne 710 System (BioRad). The proteins optical density ratios were calculated. The ratio of phosphorylated and non-phosphorylated protein was calculated.

4.5.

Statistical analysis

The data of immunoreactive cells and cell density were analyzed using an unpaired, two-tailed Student's t-test. The results are expressed as mean ± SEM. The data of immunoreactivity of the proteins were analyzed using an unpaired, two-tailed Student's t-test, with the immunoreactivity of the proteins expressed as a percent of control ± SEM.

Acknowledgments This study was supported by EU FP6 grant LSHM-CT-2005512012 (Promemoria) and Estonian Science Foundation Grant 6504, the European Regional Development Fund and the Archimedes Foundation.

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