Male imprinting control region (ICR) mice (23~25 g body weight) were obtained from Samtako (Korea). The mice were housed in a controlled environment and provided with ad libitum food and water. All animal-related procedures were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Chungnam National University (CNU-00151). We used a previously described procedure with minor modifications [17,18]. Briefly, 5 gm/ml of KA (Sigma, MO, USA) was prepared in sterile 0.1 M phosphate-buffered saline (PBS; pH 7.4). KA was injected at right lateral cerebral ventricle (anteroposterior (AP), −0.4 mm; mediolateral (ML), 1 mm; dorsoventral (DV), −2.3 mm relative to bregma) using a 50-µl Hamilton microsyringe fitted with a 26 G needle inserted to a depth of 2.4 mm (0.1 µg/5 µl in PBS, i.c.v.). Control mice received an equal volume of saline. Mice were allocated into KA-injected animals (n=6~8 per group) and saline-injected control animals (n=6~8/group). After the injection, the needle remained in place for an additional 5 min before being slowly retracted. The mice were monitored for 6 hours after KA treatment to determine the onset time of seizures. The severity of the seizures was classified into 5 stages: stage 1, facial movements; stage 2, head nodding and myoclonic twitching; stage 3, forelimb clonus with lordotic posture; stage 4, forelimb clonus with reared posture; and stage 5, tonic-clonic seizures without postural control [14,19]. The mice showing at least stage 3 seizures were considered positive for seizure onset. At 1, 3, and 7 days after KA or saline injection, mice were anesthetized using our established protocol [17,18]. Frozen coronal sections (40 µl thick) were obtained using a Leica cryostat (CM3050, Deerfield, IL, USA).

Polyclonal antisera (p-CTMP) that recognize specific phosphorylation sites were raised against PRPELRSFSSEEVILKDC (Ser-37; 29–46 aa), where the phosphorylated amino acids are underlined. Furthermore, antisera were prepared by simultaneously immunizing with the phosphoserine peptide. The phospho-peptides were coupled with Keyhole-Limpet hemocyanin and injected into rabbits. After purification by Protein A-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ) chromatography, the resultant antibodies were affinity-purified using antigenic peptides coupled to Affi-Gel 10 or 15 (Bio-Rad, Hercules, CA). All procedures were performed at 4℃.

Parallel free-floating sections were blocked with endogeneous peroxidase with 1% H₂O₂ in PBS, followed by treatment with blocking buffer (0.3% Triton X-100 and 1% fetal bovine serum [FBS] in PBS for 30 min). Samples were then incubated with primary anti-CTMP (1:100, Santa Cruz Biotechnology, CA, #sc-390353,) overnight. Immunohistochemical staining of the tissue sections was performed using the avidin–biotin peroxidase complex (ABC) method described previously [20,21]. Simultaneous staining of antigens for the double immunofluorescent experiments were performed by using Cy™3-conjugated anti-rabbit IgG (Amersham, UK) for pCTMP and p-CREB (Ser133, Upstate Biotechnology, Danvers, MA, #31554) and Cy™2-conjugated anti-mouse IgG (Amersham Pharmacia Biotech) for glial fibrillar acidic protein (GFAP, 1:1000, #AM020, Biogenex, San Ramon, CA). Nucleus staining was performed with DAPI. Axiophot microscope (Carl Zeiss, Germany) was used for the analysis of double-stained sections.

Rat primary cerebral astrocytes were purified from neonatal rats according to standard procedures [20]. Sprague-Dawley rat pups (postnatal day 1, P1) (Samtako, Korea) were decapitated in an ice-chilled dish, and the brains were harvested. After removal of the meninges, the cerebral cortex was dissected and dissociated in dissection media. After centrifugation, the cells were seeded into poly-L-lysine-coated T75 flasks and maintained in Minimal Essential Medium (MEM)-based growth media. After 7 days, the flasks were agitated on an orbital shaker, allowing for removal of the non-adherent oligodendrocytes and microglial cells. The flasks were then detached with trypsin and expanded in Dulbecco's Modified Eagle Medium (DMEM)-based astrocyte media. For lipopolysaccharide (LPS)/interferon-γ (IFN-γ) treatment, primary astrocytes were trypsinized and seeded at 70% confluence in a 60 mm dish. Then cells were further incubated for 24 hrs prior to 4 hrs of serum-starvation and stimulated with LPS (Escherichia coli 026:B6, Sigma, 100 ng/ml)/ IFN-γ (0.5 µg/ml, Sigma) for the indicated times.

For western blot, cell scrappers were used to collect cultured astrocytes PRO-PREP reagent (Intron Biotechnology, Sungnam, Korea) with a protease inhibitor cocktail (Sigma P5726, location?) was used as the lysis buffer in order to solubilize the collected cell pellet. Protein content was normalized for 30 µg of the total cellular fraction. Each sample was then separated using 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transblotted onto nitrocellulose membranes. The blot was probed with primary antibodies, p-CTMP, p-AKT (T308), CTMP, p-CREB, and β-actin antibodies (Sigma, USA) in blocking solution. Membranes were washed in TBST (3 times, 10 min each). Peroxidase (1:2000 dilution) (Vector, location?) was used for secondary antibody staining. Samples were washed 3 times then detected for immunolabeled proteins by chemiluminescence using a Supersignal ECL kit (Pierce Chemical, Rockford, IL) and Biomax Light-1 films (Kodak, USA).

Western blot was quantified by using ImageJ software (NIH), and a densitometer quantitated the relative intensity (area density) of the bands of interest. The background value was subtracted from a blank band. The results were calculated as the ratio change from the corresponding control bands. Data are presented as means±S.D. of the three independent experiments. Student's t test (SPSS version 12.0 software, SPSS Inc.) was used for the results analysis where p<0.05 (^*) was considered significant, and p<0.01 (^**), p<0.001 (^***) were considered highly significant.

The intracerebroventricular (i.c.v.) KA injection is a commonly used excitotoxicity model that promotes seizures and selective hippocampal cell death in mice [22,23]. Previously, we showed a pyramidal cell loss and glial activation in the hippocampus of this ICR mice model [18,20]. In those studies, a typical loss of pyramidal neurons in the hippocampal CA3 regions was apparent on day 1 post-injection of KA, and glial activation had increased from day 1 to day 3 and return to normal at day 7. In order to investigate the putative involvement of CTMP in KA-induced hippocampus excitotoxicity, CTMP expression in the ipsilateral hippocampus of KA-treated mice were monitored. In control mice, there was a weak signal for CTMP in parts of the hippocampus, including CA1, CA3 and the DG (Fig. 1A, E and I). However, CTMP was distinctly increased throughout the hippocampus at 3 days after KA treatment (Fig. 1C, G and K). Slides at 3 days revealed the morphology of star-shaped glial-like cells, detected by the anti-CTMP antibody, which is newly appeared in association with brain injuries (Fig. 1K). To further confirm the cell type in which CTMP was found, we employed double staining with the anti-CTMP and anti-GAFP (astrocytic marker) primary antibodies. CTMP was found to be localized to astrocytic nuclei, indicating that CTMP expression increased in the activated astrocytes in the hippocampi of KA-treated mice (data not shown). To investigate the role of phosphorylation of CTMP on Akt signaling pathway, we examined p-CTMP expression in the KA-treated mice hippocampus. The results showed that KA treatment induced an increase in p-CTMP expression in the astrocytes of hippocampus at 1 day after KA treatment (Fig. 2). As shown in Fig. 1 and 2, CTMP and p-CTMP occurred in astrocytes, indicating that CTMP expression and its phosphorylation predominantly occurred in activated astrocytes in the KA-treated mice hippocampus.

CTMP is known as a negative regulator of the Akt pathway and increased CTMP expression lead to decreased phosphorylation on both Ser473 and Thr308 residues of Akt [15]. In contrary, CTMP is released from Akt via phosphorylation of CTMP by a currently unidentified kinase which allows Akt to be phosphorylated by PDK1 and PDK2 at Thr308 and Ser473, respectively [24]. Since both CTMP phosphorylation and CTMP upregulation occurred specifically in astrocytes from KA-treated mice (Fig. 1, 2), the CTMP regulation on Akt activity in astrocyte activation by LPS/IFN-γ treatment was monitored in primary astrocytes. LPS/IFN-γ-treatment of primary astrocytes promoted p-CTMP and Akt phosphorylation (T308) at 1 h (Fig. 3A, top panel). In addition, CTMP and p-CREB increased in primary astrocytes at 6 hrs after LPS/IFN-γ-treatment (Fig 3A. lower panel). Double immunofluorescent analysis with p-CREB further confirmed that phosphorylation of CREB increased in astrocytes in the hippocampus after KA treatment (Fig. 3B). Taken together, this data indicates that phosphorylation of CTMP may regulate Akt/CREB activation and CTMP expression in astrocytes.