The knock-in transgenic (TG) mice overexpressing SRG3 were provided by Dr. R.H. Seong (Seoul National University). These mice are heterozygous for overexpressed SRG3 and produced and maintained in FVB background. The mouse SRG3 gene is designed to be overexpressed in neurons of forebrain regions by mouse CaMKII (calcium/calmodulin-dependent protein kinase II) α promoter (11.5 kb) (Benson et al., 1992; Jacobs et al., 1993; Mayford et al., 1996). The transgenic offspring was generated by crossing heterozygous transgenic mice with wild type FVB mice and genotyped by PCR using sense primer 5'-GACTA GACCA AACAT CTACC TC-3', antisense primer 5'-GTCAA CTGAG CGACT TGGAT C-3'. Mice were bred and maintained under pathogen-free conditions, and experiments were performed in accordance with institutional guidelines of Hanyang University Veterinary Committee. To evaluate the proliferation in subgranular zone (SGZ) of adult hippocampus, 5-bromo-2'-deoxyuridine (BrdU) (100 mg/kg bodyweight, Sigma-Aldrich) was daily administered intraperitoneally to twenty week-old male TG mice for 3 days and then animals were sacrificed 2 h after the last injection.

Immunoblotting followed conventional method. Briefly, hippocampal and cortical tissues were immediately collected from an isolated brain on ice after mice were sacrificed by cervical translocation. Tissues were homogenized using a Dounce homogenizer and lysed in RIPA buffer (150 mM NaCl, 10 mM Tris, 0.1% SDS, 1% Triton X-100, 5 mM EDTA, 1% Deoxycholate) including protease inhibitor cocktail (Sigma-Aldrich)/phosphatase inhibitor cocktail (Sigma-Aldrich). The kinds and dilutions of primary antibodies are as follows; anti-BAF155 (1:500, Santa Cruz Biotechnology Inc), anti-phospho-CaMKII (Thr286) (1:1,000; Cell Signaling Technology), anti-CaMKII (1:500, Cell Signaling Technology), anti-β-actin (GeneTex), anti-p-Trk (1:400, Cell Signaling Technology), anti-TrkB (1:600, Cell Signaling Technology), anti-p-ERK1/2 (1:1,000, Cell Signaling Technology), anti-ERK1/2 (1:1,000, Cell signaling Technology), anti-p-CREB (Ser133) (Abcam Inc, Cambridge, MA, USA), anti-CREB (1:500, Cell Signaling Technology), anti-BDNF (1:500, SantaCruz Biotechnology), anti-VEGF (1:200, SantaCruz Biotechnology), anti-Angiopoietin-1 (1:200, SantaCruz Biotechnology), anti-Tuj1 (1:1,000, Covance, Berkeley, CA, USA), anti-Brg1 (1:1,000, Sigma-Aldrich).

Sample tissues were collected in the same way as mentioned in immunoblotting and homogenized in Trizol reagent (Life Technologies Inc, Rockville, MD, USA). One µg of total RNA was reversetranscribed using SuperScript II reverse transcriptase (Invitrogen). The following PCR primers were employed; sense 5'-CTGCATCTTCGATCACC TCA-3', antisense 5'-CTCCCTTCCAGCACTAGCAC-3' for mouse SRG3; sense 5'-CGGAACCGC TCATTGCC-3', antisense 5'-ACCCACACTGTGCCCATCTA-3' for β-actin sense.

After anesthetized with Ketamine/Xylazine cocktail, animals were intracardially perfused with cold normal saline and, in turn, ice-cold 4% paraformaldehyde in PBS. Isolated brains were postfixed in 4% paraformaldehyde in PBS overnight at 4℃, and then fully dehydrated in 30% sucrose in PBS. Slices were prepared as 30-µm-thick. For immunoperoxidase staining, free-floating sections were first treated with 1% H₂O₂ for 15 min and washed three times with PBS. After incubated in blocking buffer (0.3% Triton X-100/5% normal serum in PBS) for 1 h, they were placed in primary antibody solution overnight at 4℃. Primary antibodies used are goat anti-BAF155 antibody (1:200, Santa Cruz Biotechnology), anti-p-CREB (Ser133) (1:200, Abcam Inc, Cambridge, MA, USA), and anti-BDNF (1:500, SantaCruz Biotechnology). Biotinylated anti-goat IgG antibody (Vector Laboratories, Burlingame, CA, USA) was used as a secondary antibody. Immunoreactivity was detected by Vectastain ABC Kit (Vector Laboratories) and DAB peroxidase substate kit (Vector Laboratories). For BrdU immunofluorescent staining, sections were incubated in 50% formamide/50% 2X SSC buffer (0.3 M NaCl and 0.03 M sodium citrate) at 65℃ for 2 h and rinsed with 2X SSC buffer for 10 min. They were then incubated for 30 min in 2 N HCl at 37℃ and rinsed in 0.1 M boric acid (pH 8.5) for 15 min. After PBS wash, they were incubated in blocking buffer for 1 h and rat anti-BrdU antibody (1:200, Abcam) solution overnight at 4℃. For Tuj-1 (1:400, Covance), PSA-NCAM (1:100, Chemicon International), NeuN (1:500, Chemicon International), MAP2 (1:400, Sigma-Aldrich) and Dcx (1:200, Santa Cruz Biotechnology), the incubation in formamide/2X SSC buffer and 2 N HCl was skipped. Cy3-conjugated anti-IgG (Jackson ImmunoResearch, West Grove, PA, USA) and Alexa Fluor 488-conjugated anti-IgG (Molecular Probes, Eugene, OR, USA) were used as secondary antibody. The samples were mounted with Vectashield (Vector Laboratories).

To confirm whether SRG3 overexpression transgenic mice are working as genetically designed to be driven by CaMKIIα promoter, which expresses in postmitotic neurons in forebrain regions like neocortex, hippocampus and amygdala (Benson et al., 1992; Jacobs et al., 1993), SRG3 TG mice were examined to see forebrain-specific overexpression of SRG3. The gross brain morphology and size of 20-week-old male SRG3 TG was not different from that of wild type (Fig. 1A), suggesting that overall development of brain is not affected by the overexpression of SRG3. Both hemispheres were symmetrical, and the size of cerebellum and olfactory bulb where CaMKIIα promoter is not activated also looks similar in SRG3 TG and wild type (wt) animals. The normal development of neocortex and the hippocampal subregions like dentate gyrus (DG), CA3 and CA1 of SRG3 TG mouse was confirmed by DAPI labeling on a coronal section at postbregma -2.0 mm (Fig. 1A). The expression of SRG3 was evaluated at the levels of mRNA and protein. Real-time RT-PCR showed the forebrain specific overexpression of SRG3 gene (Fig. 1B, hippocampus, 2.6-fold increase; cortex, 2.4-fold increase; cerebellum, 1.3-fold increase; respectively compared with wt, n=1). The SRG3 protein level revealed by western blotting using antiserum against BAF155, a human homologue of SRG3, also significantly increased in SRG3 TG mice compared to in wild type (mean±SEM; hippocampus, 143.7±5% vs wild; cortex, 137.9±5.6% vs wild, ^*p<0.05 respectively, n=3 per group) (Fig. 1C). In order to compare the expression lelvel of SRG3 protein in individual neurons, immunoperoxidase acativity against SRG3 was examined in brain slices from both groups. The SRG3 signal was detected in the nuclei (Fig. 1D inset) and it displayed more intense signals in SRG3 TG mice forebrain region than in wt (Fig. 1D). These results consistently demonstrate that the genetic manipulation for a SRG3 overexpression TG mouse is reliablely working, showing an increased expression pattern for SRG3 mRNA and protein in forebrain neurons.

To determine whether the overexpression of SRG3 causes an altered pattern in the proliferation of neural progenitor cells, the number of newly generated progenitor cells in SGZ was examined by the incorporation of BrdU. Although CaMKIIα promoter does not operate in proliferating cells, the neural progenitor cells in the DG of adult hippocampus can be affected indirectly by postmitotic neurons (Chen et al., 2007) overexpressing SRG3 gene by CaMKIIα promoter. BrdU immunopositive (BrdU+) cells were counted within SGZ throughout the DG of both genotypes (Fig. 2A). Some BrdU+ cells were found in clusters like those in the previous reports (Yagita et al., 2001; Zhu et al., 2005; Hodge et al., 2008). The total number of BrdU+ cells located in the SGZ (mean±SEM; wt, 1,099±63/mm³ vs. SRG3 TG, 1,024±59/mm³, p>0.1, n=4 per group) was not statistically different between wt and SRG3 TG mice (Fig. 2B), indicating that the proliferation was not affected by the overexpression of SRG3.

Cell proliferation in the subventricular zone (SVZ) of a lateral ventricle was also examined (Gould, 2007). Though CaMKIIα promoter operates only in matured neurons, a possible effect of the overexpression of SRG3 on the proliferation pattern of SVZ was needed to be checked when considering of SVZ as another important niche for adult neurogenesis. However, no difference in the proliferation rate in SVZ was found in both genotypes (Fig. 2C).

To determine whether early neuronal development is normally organized in SRG3 TG mice, postomitotic differentiation in a hippocampal region was immunostained with early neuronal markers. During postmitotic and neuronal differentiation stage in adult hippocampal neurogenesis, most newborn neurons are destined to die, but only survived neurons mature to integrate into a functional neural circuit (Tashiro et al., 2007). Previous studies reported that cells committed to a neuronal lineage start to express doublecortin (Dcx) (Francis et al., 1999) and the polysialylated embryonic form of the neural cell adhesion molecule (PSA-NCAM), right after they exit out of the cell cycle (Fukuda et al., 2003). The total number of Dcx immunoreactive (Dcx+) cells in the hippocami of SRG3 TG mice was not significantly changed compared to that of wt (mean number±SEM: wt, 9,974±450/mm³; SRG3 TG, 9,133±701/mm³, p>0.1, n=4 per group), suggesting that neuronal differentiation of neural progenitor cells in the hippocampus of adult SRG3 TG mice seems normal during early postmitotic period (Fig. 3A). As Dcx expression is temporally overlapped with PSA-NCAM expression on the time course of development, the immunoreactivity of PSA-NCAM+ cells on randomly chosen brain slices from both genotypes cells was compared (Nacher et al., 2001). It turns out that the number and the signal intensity of PSA-NCAM+ cells was apparently similar to result of Dcx immunostaining and there is no difference between SRG3 TG and wt groups.

In order to know what effect the overexpressed SRG3 on mature neurons has on the maturation of the hippocampi and cortex, Tuj-1 was used as a neuroblast marker as well as a fully differentiated neuronal marker to examine SRG3 TG mice. Neuron-specific class III β-tubulin (Tuj-1) has been found to be expressed in newly generated immature postmitotic neurons (Menezes and Luskin, 1994) and in mature neurons (Ambrogini et al., 2004) (Fig. 3C). Tuj-1 was detected in somata and neurites of neurons within cortex, CA1 and DG. Especially, in DG, Tuj-1 was detected in the somata of almost all granule cells and found at the neurites of some granule cells reaching the hippocampal fissure. The distribution and immunoreactivity of Tuj-1 in cortex, CA1 and DG was indistinguishable between SRG3 TG and wt animals, suggesting that the neuronal maturation in adult hippocampal neurogenesis as well as cortical neuronal development appear normal in SRG3 TG mice.

To see whether overexpressed SRG3 affects the late neuronal differentiation process and the survival of neuronal lineage-committed cells during the late stage of adult hippocampal neurogenesis, microtubule-associated protein 2 (MAP2) and NeuN were immunolabeled, as MAP2 and NeuN have been known as mature neuronal markers (Menezes and Luskin, 1994). The signal was detected at dendrites and somata of neurons (Fig. 4A) as reported previously (Biranowska et al., 2000; Jalava et al., 2007). Though there were some cells in SGZ missing MAP2 immunoreactivity, they might be immature or proliferating cells. The distribution and immunoreactivity of MAP2 immunopositive (MAP2+) cells in the cortex and the hippocampus was not significantly different in SRG3 TG and wt animals (Fig. 4A). In addition, the immubolabeling of NeuN, a nuclear protein for labeling the nuclei of mature neurons (Lind et al., 2005), displayed a similar pattern in SRG3 TG and wt mice (Fig. 4B), suggesting that the late neuronal differentiation process during adult hippocampal neurogenesis and the neuronal survival remains normal in SRG3 TG mice.

Though the immunostaining with MAP2 and Tuj-1 results in no impaired pattern of the survival of newly generated hippocampal neurons by the overexpressed SRG3, this might not mean that the neuronal maturation is completely normal. To determine whether the neuronal survival is not altered in a subtle manner by the overexpression of SRG3, CREB activation, which is a critical step for survival of newborn neurons in a hippocampus (Nakagawa et al., 2002), was examined using western blotting and immunolabeling. ERK1/2 and CaMKII, critical mediators in activating CREB, were expressed and activated to a similar degree (Fig. 5A, pERK1/2, 104±6% vs wt, p>0.05; pCaMKII, 97±2% vs wt, p>0.05), and similarly CREB activation was not different between two groups (Fig. 5A, pCREB, 106±3% vs wt, p>0.05). Moreover, the expression of BDNF regulated by phosphorylated CREB and the activation of TrkB, a receptor for BDNF, were not significantly different in both groups (Fig. 5A: BDNF, 103±3% vs. wt, p>0.05; pTrkB, 106±5% vs wt, p>0.05), which is consistent with the immunolabeling of each marker on tissues, implying that neuronal survival and the survival signaling is not affected by the overexpression of SRG3 in mature neurons.

To see whether the expression of VEGF and angiopoietin1 was altered by the overexpressed SRG3, the western blotting was performed with using hippocampal homogenates from each genotype, because vascular endothelial growth factor (VEGF) and angiopoietin1 are all angiogenesisrelated and were reported to be linked to adult neurogenesis (During and Cao, 2006; Warner-Schmidt et al., 2008). Recently, SRG3 has been reported to be required for angiogenesis and visceral endoderm development in the yolk sac, and the expression of angiogenesis-related genes, includeing Angiopoietin1, Tie2, EphrinB2, Ihh and Notch1, was markedly reduced in SRG3-/-Tg+ yolk sacs (Han et al., 2008). The expression of VEGF and angiopoietin1 was not different in both genotypes (Fig. 5A: VEGF, 104±3% vs wt, p>0.05; angiopoietin1, 102±3% vs wt, p>0.05), unlike the reduced expression of angiogenesis related genes by the depletion of SRG3.

To overcome the possible limitation of the western blotting missing the subtle change of the altered expression level of specific molecules which are very small portion of the whole hippocampus, the activation or expression of BDNF and CREB was examined by immunofluorescent and immunoperoxidase staining at a single cell level (Fig. 5B, C). BDNF signal was not strongly detected at somata and dendritic arbors of hippocampal neurons from both groups and rather more intensely observed in neurites of cortical neurons (Fig. 5B). The phosphorylated CREB was detected in nuclei of cells within SGZ and across cortical layers (Fig. 6C). Taken together, a line of evidence from western blotting and immunostaining with phosphorylated-CREB and BDNF clearly demonstrate that overexpression of SRG3 makes no significant impact on the expression and activation of intracellular signaling molecules involved in the survival of newborn neurons in adult hippocampus.