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Impaired social facilitation was reported in autism spectrum disorder (ASD) children. However, behavioral analysis methods of social facilitation for ASD model have not been reported. We developed a novel breeding home cage for social facilitation. Voluntary exercise of more social C57BL/6 J mice was significantly increased in the presence of observer mouse compared to that in the absence of observer mouse. In contrast, the presence of observer mouse did not affect voluntary exercise of less social BALB/cCrSlc mice. These suggest that BALB/cCrSlc mice, a mouse model of ASD, exhibited impaired social facilitation. Our method would provide novel clues for ASD pathophysiology.

New neurons, continuously added in the adult olfactory bulb (OB) and hippocampus, are involved in information processing in neural circuits. Here, we show that synaptic pruning of adult-born neurons by microglia depends on phosphatidylserine (PS), whose exposure on dendritic spines is inversely correlated with their input activity. To study the role of PS in spine pruning by microglia in vivo, we developed an inducible transgenic mouse line, in which the exposed PS is masked by a dominant-negative form of milk fat globule-EGF-factor 8 (MFG-E8), MFG-E8D89E. In this transgenic mouse, the spine pruning of adult-born neurons by microglia is impaired in the OB and hippocampus. Furthermore, the electrophysiological properties of these adult-born neurons are altered in MFG-E8D89E mice. These data suggest that PS is involved in the microglial spine pruning and the functional maturation of adult-born neurons. The MFG-E8D89E–based genetic approach shown in this study has broad applications for understanding the biology of PS-mediated phagocytosis in vivo.

// Shotaro Ando 1 , Jun-ichi Kawada 1 , Takahiro Watanabe 2 , Michio Suzuki 1 , Yoshitaka Sato 2 , Yuka Torii 1 , Masato Asai 3 , Fumi Goshima 2 , Takayuki Murata 2 , Norio Shimizu 4 , Yoshinori Ito 1 , Hiroshi Kimura 2 1 Departments of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan 2 Departments of Virology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan 3 Departments of Pathology, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya 466-8550, Japan 4 Center of Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan Correspondence to: Jun-ichi Kawada, email: kawadaj@med.nagoya-u.ac.jp Keywords: tofacitinib, EBV, lymphoma, cell-cycle arrest Received: June 13, 2016 Accepted: October 03, 2016 Published: October 08, 2016 ABSTRACT Epstein-Barr virus (EBV) infects not only B cells, but also T cells and natural killer (NK) cells, and is associated with T or NK cell lymphoma. These lymphoid malignancies are refractory to conventional chemotherapy. We examined the activation of the JAK3/STAT5 pathway in EBV-positive and -negative B, T and NK cell lines and in cell samples from patients with EBV-associated T cell lymphoma. We then evaluated the antitumor effects of the selective JAK3 inhibitor, tofacitinib, against these cell lines in vitro and in a murine xenograft model. We found that all EBV-positive T and NK cell lines and patient samples tested displayed activation of the JAK3/STAT5 pathway. Treatment of these cell lines with tofacitinib reduced the levels of phospho-STAT5, suppressed proliferation, induced G1 cell-cycle arrest and decreased EBV LMP1 and EBNA1 expression. An EBV-negative NK cell line was also sensitive to tofacitinib, whereas an EBV-infected NK cell line was more sensitive to tofacitinib than its parental line. Tofacitinib significantly inhibited the growth of established tumors in NOG mice. These findings suggest that tofacitinib may represent a useful therapeutic agent for patients with EBV-associated T and NK cell lymphoma.

A long-standing paradigm posits that hypothalamic corticotropin-releasing hormone (CRH) regulates neuroendocrine functions such as adrenal glucocorticoid release, whereas extra-hypothalamic CRH has a key role in stressor-triggered behaviors. Here we report that hypothalamus-specific Crh knockout mice (Sim1CrhKO mice, created by crossing Crhflox with Sim1Cre mice) have absent Crh mRNA and peptide mainly in the paraventricular nucleus of the hypothalamus (PVH) but preserved Crh expression in other brain regions including amygdala and cerebral cortex. As expected, Sim1CrhKO mice exhibit adrenal atrophy as well as decreased basal, diurnal and stressor-stimulated plasma corticosterone secretion and basal plasma adrenocorticotropic hormone, but surprisingly, have a profound anxiolytic phenotype when evaluated using multiple stressors including open-field, elevated plus maze, holeboard, light-dark box and novel object recognition task. Restoring plasma corticosterone did not reverse the anxiolytic phenotype of Sim1CrhKO mice. Crh-Cre driver mice revealed that PVHCrh fibers project abundantly to cingulate cortex and the nucleus accumbens shell, and moderately to medial amygdala, locus coeruleus and solitary tract, consistent with the existence of PVHCrh-dependent behavioral pathways. Although previous, nonselective attenuation of CRH production or action, genetically in mice and pharmacologically in humans, respectively, has not produced the anticipated anxiolytic effects, our data show that targeted interference specifically with hypothalamic Crh expression results in anxiolysis. Our data identify neurons that express both Sim1 and Crh as a cellular entry point into the study of CRH-mediated, anxiety-like behaviors and their therapeutic attenuation.

Neurodevelopmental disorders, including intellectual disability and autism spectrum disorder, are often caused by de novo autosomal dominant mutations. While mouse models are frequently used to investigate these disorders, the genetic background sometimes affects the appearance or severity of mutant phenotypes. In a previous report, we developed a system to produce de novo heterozygous mutant mice using the Cre-LoxP system without the need to maintain the heterozygous mutant line itself (Takagi et al. 2015). To further verify the applicability of the de novo mutation system in sperm, we used this system to produce a mouse model for Rubinstein-Taybi syndrome, using a Cbp heterozygous mutant, which has been reported to be difficult to maintain on a C57BL/6 background. Here, we show that de novo Cbp- loss-of-function heterozygous mutant mice with a C57BL/6 background, present with a clear craniofacial phenotype and reduced locomotor activity in the open field test, which was not observed in the loss-of-function of Cbp heterozygous mutant line mice with a mixed genetic background, but was observed in the dominant negative Cbp heterozygous mutant line with a mixed genetic background. Meanwhile, the de novo heterozygous Cbp mutant mice still showed great variability in survival rates despite their inbred background. These results further confirmed that the de novo mutation system used in germ cells is effective for stable production and analysis of an autosomal dominant disorder mouse model, which is often difficult to maintain as a mutant mouse line.

In gastric cancer, the non‐canonical Wnt signaling pathway is activated by Wnt5a, which has a critical role in disease outcome. Previous studies have shown that Wnt5a mediates the expression of the extracellular matrix protein laminin γ2 through Rac and JNK activation to promote gastric cancer progression. However, the mechanism of this regulatory pathway has not been completely addressed. The scaffold protein Dvl is a major component of the Wnt signaling pathway. Here, we show that Dvl‐associating protein with a high frequency of leucine residues (Daple) mediates Wnt5a‐induced laminin γ2 expression. Immunohistochemical analysis showed marked expression of Daple in advanced clinical stages of gastric cancer, where it highly correlated with Wnt5a/b and laminin γ2 expression, the depth of wall invasion, and the frequency of lymph node metastasis. In cultured cancer cells, Daple depletion led to the suppression of Wnt5a‐induced Rac and JNK activation, laminin γ2 expression, and cell migration and invasion. Accordingly, Daple depletion also suppressed liver metastasis in a mouse xenograft model of gastric cancer. These results suggest that the non‐canonical Wnt signaling pathway contributes to gastric cancer progression at least in part via Daple, which provides a new therapeutic opportunity for the treatment of the disease.

The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is phosphorylated by both receptor and non-receptor tyrosine kinases at tyrosine 1798. Omori et al. developed site- and phosphorylation status-specific antibodies against human girdin at tyrosine-1798 (pY1798), which specifically bind to phosphorylated tyrosine-1798, but not to unphosphorylated tyrosine-1798. pY1798 antibodies have been used to specifically label tuft cells (TCs) that are present in mammalian gastrointestinal tissues, but the function of these cells is unclear. This protocol allows the robust visualization of TCs in the jejunum using pY1798 antibodies and immunofluorescence. To ensure successful and simple TC visualization, this protocol includes two histological techniques: production of free-floating cryosections from gelatin-filled jejunum tissue, and low-temperature antigen retrieval at 50 °C for 3 h. Filling the jejunum with gelatin maintains the shape of free-floating sections throughout the staining procedure, whereas low-temperature antigen retrieval ensures robust signals from TCs. Successful use of this protocol results in pY1798 staining of TCs distributed from villus tip to crypt. Stained TCs have a spool-shaped soma and fluorescent signals condense at the lumenal tip, which corresponds to the protruding 'tuft.' Phalloidin staining colocalized with pY1798-positive TCs at the thickened brush border, and corresponds to a rootlet mass extending from the TC tuft. This protocol could be used to examine TCs in human biopsy samples collected with gastrointestinal endoscopes. Furthermore, TCs were recently reported to accumulate following parasite infection in mice, suggesting that this protocol could have applications for diagnosis of parasite infections in the human gut.

Amino acid signaling mediated by the activation of mechanistic target of rapamycin complex 1 (mTORC1) is fundamental to cell growth and metabolism. However, how cells negatively regulate amino acid signaling remains largely unknown. Here, we show that interaction between 4F2 heavy chain (4F2hc), a subunit of multiple amino acid transporters, and the multifunctional hub protein girders of actin filaments (Girdin) down-regulates mTORC1 activity. 4F2hc interacts with Girdin in mitogen-activated protein kinase (MAPK)- and amino acid signaling–dependent manners to translocate to the lysosome. The resultant decrease in cell surface 4F2hc leads to lowered cytoplasmic glutamine (Gln) and leucine (Leu) content, which down-regulates amino acid signaling. Consistently, Girdin depletion augments amino acid-induced mTORC1 activation and inhibits amino acid deprivation–induced autophagy. These findings uncovered the mechanism underlying negative regulation of amino acid signaling, which may play a role in tightly regulated cell growth and metabolism., Author summary The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master regulator of cell growth, which senses several extracellular signals, such as growth factors and nutrient levels, to coordinate cell metabolism. The activation of mTORC1 by amino acids requires many proteins such as Rag GTPase, GATOR, and Ragulator. However, how cells negatively regulate amino acid signaling remains largely unknown. In this study, we revealed that an endocytosis-related protein called Girdin negatively regulates amino acid–induced mTORC1 activation via the formation of a complex with 4F2 heavy chain (4F2hc), a subunit of multiple amino acid transporters. We show that Girdin/4F2h complex formation requires growth factor-induced Girdin phosphorylation and amino acid–induced 4F2hc ubiquitination. We also find that the complex promotes the internalization of 4F2hc from the plasma membrane to the lysosomes. The subsequent decrease of 4F2hc in the cell surface results in a lower cytoplasmic glutamine and leucine content, which then down-regulates amino acid–induced mTORC1 activation. These findings uncover the mechanism underlying negative regulation of mTORC1 signaling, which may play a role in tightly regulated cell growth and metabolism.

The mammalian protein Girdin interacts with several key molecules such as actin, and it functions as a regulator of the cytoskeleton. Silencing of Girdin mRNA results in defective migration in a variety of cultured cells. Moreover, knockout of Girdin causes phenotypes related to defective migration, including hypoplasia of olfactory bulbs and a widened rostral migratory stream (RMS) in mice. To elucidate the molecular basis underlying cellular migration, we generated site- and phosphorylation state-specific antibodies against human Girdin peptides carrying four putative phosphorylation sites (serine1386 [S1386], S1416, tyrosine1764 [Y1764] and Y1798) that had been identified by mutagenesis analyses or mass spectrometric studies. We found that these residues were phosphorylated in an epidermal growth factor (EGF)-dependent manner. Among the four antibodies we developed, the antibody that targeted Girdin when phosphorylated at Y1798 (pY1798) worked well for immunohistochemistry of paraffin-embedded tissues as well as for cultured cells. Immunocytochemistry of HEK293FT cells transfected with an EGF receptor expression plasmid exhibited punctate signals with pY1798. These signals colocalized with those of endocytosed EGF receptors after EGF stimulation. Signals from pY1798 were also observed on lamellipodia, filopodia, focal adhesion and stress fibers in NIH3T3 cells under conventional culture conditions. Immunohistochemistry of paraffin-embedded mouse brain at P14 using anti-pY1798 antibody displayed signals at the hilum-side (internal side) of the dentate gyrus of the hippocampus, the RMS, the accessory olfactory bulb and the olfactory bulb in which Girdin expression was detected. Primary culture of RMS neurons showed punctate signals of pY1798 at the tips of leading processes as well as in the cytoplasm, whereas no signals were observed when neurons were treated with Src inhibitor, PP2. Our data revealed the changes in the phosphorylation status of Y1798 in Girdin when it associated with migration-related structures in vitro and in vivo.

Adult Cebpb KO mice incisors present amelogenin-positive epithelium pearls, enamel and dentin allopathic hyperplasia, fewer Sox2-positive cells in labial cervical loop epitheliums, and reduced Sox2 expression in enamel epithelial stem cells. Thus, Cebpb acts upstream of Sox2 to regulate stemness. In this study, Cebpb KO mice demonstrated cementum-like hard tissue in dental pulp, loss of polarity by ameloblasts, enamel matrix in ameloblastic layer, and increased expression of epithelial-mesenchymal transition (EMT) markers in a Cebpb knockdown mouse enamel epithelial stem cell line. Runx2 knockdown in the cell line presented a similar expression pattern. Therefore, the EMT enabled disengaged odontogenic epithelial stem cells to develop supernumerary teeth. Cebpb and Runx2 knockdown in the cell line revealed higher Biglycan and Decorin expression, and Decorin-positive staining in the periapical region, indicating their involvement in supernumerary tooth formation. Cebpb and Runx2 acted synergistically and played an important role in the formation of supernumerary teeth in adult incisors.

Summary Tuft cells (TCs) are minor components of gastrointestinal epithelia, characterized by apical tufts and spool-shaped somas. The lack of reliable TC-markers has hindered the elucidation of its role. We developed site-specific and phosphorylation-status–specific antibodies against Girdin at tyrosine-1798 (pY1798) and found pY1798 immunostaining of mouse jejunum clearly depicted epithelial cells closely resembling TCs. This study aimed to validate pY1798 as a TC-marker. Double-fluorescence staining of intestines was performed with pY1798 and known TC-markers, for example, hematopoietic-prostaglandin-D-synthase (HPGDS), or doublecortin-like kinase 1 (DCLK1). Odds ratios (ORs) were calculated from cell counts to determine whether two markers were attracting (OR1). In consequence, pY1798 signals strongly attracted those of known TC-markers. ORs for HPGDS in mouse stomach, small intestine, and colon were 0 for all, and 0.08 for DCLK1 in human small intestine. pY1798-positive cells in jejunum were distinct from other minor epithelial cells, including goblet, Paneth, and neuroendocrine cells. Thus, pY1798 was validated as a TC-marker. Interestingly, apoptosis inducers significantly increased relative TC frequencies despite the absence of proliferation at baseline. In conclusion, pY1798 is a novel TC-marker. Selective tyrosine phosphorylation and possible resistance to apoptosis inducers implied the activation of certain kinase(s) in TCs, which may become a clue to elucidate the enigmatic roles of TCs.

Human REV7 (also known as MAD2L2 and MAD2B) is involved in DNA repair, cell cycle regulation, gene transcription, and carcinogenesis. In this study, we evaluated the expression of REV7 in epithelial ovarian cancer (EOC) and analyzed the association between its expression and chemosensitivity in ovarian clear cell carcinoma (CCC) cells. Expression of REV7 in human EOC tissues was assessed by immunohistochemical staining. Expression was detected in the majority of EOCs (92.0%) with especially high levels of expression frequently observed in CCCs (73.5%) compared with that of non-CCCs (53.4%). Enhanced immunoreactivity to REV7 was associated with poor prognosis represented by reduced progression-free survival in advanced stage (stage II–IV) EOC as assessed using Kaplan–Meier curves and log–rank tests. The effects of REV7 knockdown on cell proliferation and chemosensitivity in CCC cells were also analyzed in vitro and in vivo. Knockdown of REV7 in CCC cells decreased cell proliferation without affecting cell cycle distribution. Additionally, the number of apoptotic cells and DNA damaged cells were increased after cisplatin treatment. In a nude mouse tumor xenograft model, inoculated REV7-knockdown tumors showed significantly reduced tumor volumes after cisplatin treatment compared with those of the control group. These findings indicate that depletion of REV7 enhances sensitivity to cisplatin treatment in CCC, suggesting that REV7 is a candidate molecular target in CCC management.

Accessory to Obesity? Melanocortin receptors are a family of cell membrane receptors that control diverse physiological functions. Mutations in the gene encoding melanocortin 4 receptor (MC4R) are a cause of familial early-onset obesity. Asai et al. (p. 275 ) studied the function of an accessory protein for MC4R signaling, MRAP2, and found that mice genetically deficient in MRAP2 develop severe obesity. Sequencing of MRAP2 in unrelated, severely obese humans revealed one individual with a clearly disruptive genetic variant, suggesting that MRAP2 mutations might also be a rare cause of human obesity. In a zebrafish model, Sebag et al. (p. 278 ) studied two paralogs of the MRAP2 accessory protein, one of which enhanced MC4R responsiveness to α–melanocyte-stimulating hormone, which regulates feeding and growth.

REV7 (also known as MAD2L2 and MAD2B) is involved in DNA repair, cell cycle regulation, gene expression, and carcinogenesis. In vitro studies show that REV7 interacts with several proteins and regulates their function. It has been reported that human REV7 is highly expressed in the adult testis by Northern blot analysis. However, the significance of REV7 in mammalian development has not been elucidated. Here, we present analyses of REV7-deficient (Rev7(-/-)) mice to clarify the significance of Rev7 in mouse development. In WT mice (Rev7(+/+)), Rev7 expression was ubiquitously observed in the embryo and confined to germ cells in the testes after birth. Rev7(-/-) mice exhibited growth retardation and a partial embryonic lethal phenotype. Mice that survived to adulthood were infertile in both sexes and showed germ cell aplasia in the testes and ovaries. Analyses of Rev7(-/-) embryos revealed that primordial germ cells (PGCs) were present at embryonic day 8.5 (E8.5). However, progressive loss of PGCs was observed during migration, and PGCs were absent in the genital ridges at E13.5. An increase of apoptotic cells was detected not only among PGCs but also in the forebrain of the Rev7(-/-) embryo, whereas cell proliferation was unaffected. Moreover, DNA damage accumulation and increased levels of histone methylation were detected in Rev7(-/-) embryos, and expression of Oct4 and Nanog was deregulated by REV7 deficiency at E8.5. These findings indicate that Rev7 is essential for PGC maintenance by prevention of apoptotic cell death in the mouse.

Resistance to platinum- and taxane-based chemotherapy is a major cause of treatment failure in ovarian cancer. Thus, it is necessary to develop a predictive marker and molecular target for overcoming drug resistance in ovarian cancer treatment. In a previous report, using an in vitro model, we found that the RET finger protein (RFP) (also known as tripartite motif-containing protein 27, TRIM27) confers cancer cell resistance to anticancer drugs. However, the significance of RFP expression in cancer patients remains elusive. In this study, we showed that RFP was expressed in 62% of ovarian cancer patients and its positivity significantly correlated with drug resistance. Consistent with clinical data, depletion of RFP by RNA interference (RNAi) in ovarian cancer cell lines, SKOV3 and HEY, significantly increased carboplatin- or paclitaxel-induced apoptosis and resulted in reduced anticancer drug resistance. In a nude mouse tumor xenograft model, inoculated RFP-knockdown ovarian cancer cells exhibited lower carboplatin resistance than control cells. These findings suggest that RFP could be a predictive marker for chemoresistance in ovarian cancer patients and also a candidate for a molecular-targeted agent.

The glial cell line-derived neurotrophic factor (GDNF)/RET tyrosine kinase signaling pathway plays crucial roles in the development of the enteric nervous system (ENS) and the kidney. Tyrosine 1062 (Y1062) in RET is an autophosphorylation residue that is responsible for the activation of the PI3K/AKT and RAS/MAPK signaling pathways. Mice lacking signaling via Ret Y1062 show renal hypoplasia and hypoganglionosis of the ENS although the phenotype is milder than the Gdnf- or Ret-deficient mice. Sprouty2 (Spry2) was found to be an antagonist for fibroblast growth factor receptor (FGFR) and acts as an inhibitory regulator of ERK activation. Spry2-deficient mice exhibit hearing loss and enteric nerve hyperplasia. In the present study, we generated Spry2-deficient and Ret Y1062F knock-in (tyrosine 1062 is replaced with phenylalanine) double mutant mice to see if abnormalities of the ENS and kidney, caused by loss of signaling via Ret Y1062, are rescued by a deficiency of Spry2. Double mutant mice showed significant recovery of ureteric bud branching and ENS development in the stomach. These results indicate that Spry2 regulates downstream signaling mediated by GDNF/RET signaling complex in vivo.

Objective: The precise mechanism whereby proinflammatory cytokines activate the hypothalamo-pituitary-adrenal axis is still unclear. We examined whether transcription factors nuclear factor (NF)-ĸB and Nurr-1 are involved in the cytokine-induced proopiomelanocortin (POMC) gene expression. Methods: The mouse corticotropinoma cell line AtT20 was treated with tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β). Real-time PCR, luciferase assay and Western blotting were conducted to assess the gene expression, promoter activity and protein expression in various conditions. Results: Intrinsic expression of NF-ĸB was confirmed by RT-PCR. An active component of NF-ĸB (p65) was upregulated in the nuclear fraction by both TNF-α and IL-1β treatment in a dose- and time-related manner. These cytokines potently stimulated the promoter activity of NF-ĸB and Nurr-1. We also found rapid upregulation of the Nurr-1 gene and protein after treatment with these cytokines. Cotreatment of the cells with either of the cytokines and corticotropin-releasing hormone resulted in additive effects. Cytokine-induced Nurr-1 transcription and Nurr-1 transcription induced by overexpression of NF-ĸB were both blunted by mutagenesis within the NF-ĸB responsive element, which implies that Nurr-1 upregulation specifically requires NF-ĸB binding to its own DNA-binding site. Proinflammatory cytokines exert positive effects on POMC gene expression, which were inhibited by pretreatment with a specific NF-ĸB inhibitor. Conclusion: These results together imply that Nurr-1 expression is a connecting point between neuroendocrine and immune systems in mediating cytokine-induced POMC gene expression.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) in culture are derived from BM stromal cells or skeletal stem cells. Whereas MSCs have been exploited in clinical medicine, the identification of MSC-specific markers has been limited. Here, we report that a cell surface and secreted protein, Meflin, is expressed in cultured MSCs, fibroblasts and pericytes, but not other types of cells including epithelial, endothelial and smooth muscle cells. In vivo, Meflin is expressed by immature osteoblasts and chondroblasts. In addition, Meflin is found on stromal cells distributed throughout the BM and on pericytes and perivascular cells in multiple organs. Meflin maintains the undifferentiated state of cultured MSCs and is downregulated upon their differentiation, consistent with the observation that Meflin-deficient mice exhibit increased number of osteoblasts and accelerated bone development. In the bone and BM, Meflin is more highly expressed in primitive stromal cells that express platelet-derived growth factor receptor α and Sca-1 than the Sca-1-negative adipo-osteogenic progenitors, which create a niche for hematopoiesis. Those results are consistent with a decrease in the number of clonogenic colony-forming unit-fibroblasts within the BM of Meflin-deficient mice. These preliminary data suggest that Meflin is a potential marker for cultured MSCs and their source cells in vivo.

P rogressive E ncephalopathy with oedema, H ypsarrhythmia and O ptic atrophy (PEHO) is a rare, neurodegenerative disorder of unknown aetiology. Nahorski et al . identify the first causative recessive mutation in CCDC88A , which encodes the actin-binding protein Girdin. The phenotype and brain anatomy of the Ccdc88a knockout mouse resemble those of human PEHO syndrome., Progressive encephalopathy with oedema, hypsarrhythmia and optic atrophy (PEHO) syndrome is a rare Mendelian phenotype comprising severe retardation, early onset epileptic seizures, optic nerve/cerebellar atrophy, pedal oedema, and early death. Atypical cases are often known as PEHO-like, and there is an overlap with ‘early infantile epileptic encephalopathy’. PEHO is considered to be recessive, but surprisingly since initial description in 1991, no causative recessive gene(s) have been described. Hence, we report a multiplex consanguineous family with the PEHO phenotype where affected individuals had a homozygous frame-shift deletion in CCDC88A (c.2313delT, p.Leu772*ter). Analysis of cDNA extracted from patient lymphocytes unexpectedly failed to show non-sense mediated decay, and we demonstrate that the mutation produces a truncated protein lacking the crucial C-terminal half of CCDC88A (girdin). To further investigate the possible role of CCDC88A in human neurodevelopment we re-examined the behaviour and neuroanatomy of Ccdc88a knockout pups. These mice had mesial-temporal lobe epilepsy, microcephaly and corpus callosum deficiency, and by postnatal Day 21, microcephaly; the mice died at an early age. As the mouse knockout phenotype mimics the human PEHO phenotype this suggests that loss of CCDC88A is a cause of the PEHO phenotype, and that CCDC88A is essential for multiple aspects of normal human neurodevelopment.

Girdin is an actin-binding protein that has multiple functions in postnatal neural development and cancer progression. We previously showed that Girdin is a regulator of migration for neuroblasts born from neural stem cells in the subventricular zone (SVZ) and the dentate gyrus of the hippocampus in the postnatal brain. Despite a growing list of Girdin-interacting proteins, the mechanism of Girdin-mediated migration has not been fully elucidated. Girdin interacts with Disrupted-In-Schizophrenia 1 and partitioning-defective 3, both of which have been shown to interact with the kinesin microtubule motor proteins. Based on this, we have identified that Girdin also interacts with kinesin-1, a member of neuronal kinesin proteins. Although a direct interaction of Girdin and kinesin-1 has not been determined, it is of interest to find that Girdin loss-of-function mutant mice with the mutation of a basic amino acid residue-rich region (Basic mut mice) exhibit limited interaction with kinesin-1. Furthermore, expression of a kinesin-1 mutant with motor defects, leads to Girdin mislocalization. Finally, consistent with previous studies on the role of kinesin proteins in trafficking a cell-cell adhesion molecule N-cadherin, Basic mut mice showed an aberrant expression pattern of N-cadherin in migrating SVZ neuroblasts. These findings suggest a potential role of Girdin/kinesin-1 interaction in the regulation of neuroblast migration in the postnatal brain.

Arginine vasopressin (AVP) is expressed in paraventricular, supraoptic, and suprachiasmatic nuclei of the hypothalamus, where transcription of the AVP gene is activated by various forms of stress, such as hyperosmolality, inflammation, and photic stimulation. In vasopressinergic neurons, the expression of the Fos/Jun family proteins is known to be rapidly induced after these stimuli as well. However, it is still unknown whether these proteins actually mediate AVP gene expression. In this study we examined in vitro the role of Fos/Jun protein in transcriptional regulation of the AVP gene using the BE(2)M17 neuroblastoma cell line. We found that 5'-promoter activity of the rat AVP gene (-803/+26) markedly increased when all combinations of the Fos/Jun family proteins were overexpressed. Coexpression of the cAMP-responsive element-binding protein-binding protein and steroid receptor coactivator-1a further enhanced the Fos/Jun-mediated transcription. Using site-directed mutagenesis and EMSA techniques, we identified an activation protein 1 (AP1)-like element (-134/-128; TGAATCA) in the AVP gene 5'-promoter region, which is the sole responsible site for the Fos/Jun-mediated transcription. We also found that 12-O-tetradecarbonyl phorbol 13-acetate stimulates AVP gene transcription partly via the AP1 site through the activation of ERK signaling. Together, these results suggest that a variety of Fos/Jun family member proteins stimulate transcription of the AVP gene through the AP1 site we identified. Furthermore, this effect may be activated by both protein kinase A and protein kinase C signaling pathways.

GHRH plays a pivotal role in the regulation of both synthesis and secretion of GH in the anterior pituitary. In this study, we examined the molecular mechanism of depolarization-induced GHRH gene transcription using the hypothalamus cell line, Gsh+/+, revealing the involvement of the transcription factor called nuclear factor of activated T cells (NFAT). GHRH, NFAT1, NFAT4, and related genes were endogenously expressed in Gsh+/+ cells and the rat arcuate nucleus, where NFAT1 and GHRH were colocalized. Cellular excitation with high potassium potently stimulated endogenous GHRH gene 5′-promoter activity as well as the NFAT-mediated gene transcription, the former being further enhanced by coexpression of NFAT. On the other hand, cyclosporin A (a calcineurin-NFAT inhibitor) or EGTA (a calcium chelator) significantly blocked the depolarization-induced GHRH gene transcription. EMSA and site-directed mutagenesis experiments showed the direct binding of NFAT at five sites of the GHRH promoter, among which the relative importance of three distal sites (−417/−403, −402/−387, −317/−301) was suggested. Finally, elimination of all five sites completely abolished the NFAT-induced GHRH gene up-regulation. Altogether, our results suggest that the transcription factor NFAT is involved in the depolarization-induced transcriptional activation of GHRH gene in the neuronal cells.

Synaptic plasticity in hippocampal neurons has been thought to represent a variety of memories. Although accumulating evidence indicates a crucial role of BDNF/TrkB/Akt signaling in the synaptic plasticity of the hippocampus, the mechanism by which Akt, a serine/threonine kinase, controls activity-dependent neuronal plasticity remains unclear. Girdin (also known as APE, GIV, and HkRP1), an actin-binding protein involved both in the remodeling of the actin cytoskeleton and in cell migration, has been identified as a substrate of Akt. Previous studies have demonstrated that deficit of neuronal migration in the hippocampus ofGirdin-deficient (Girdin−/−) mice is independent on serine phosphorylation of Girdin at S1416 (Girdin S1416) by Akt. In the present study, we focused on the role of Girdin S1416 phosphorylation in BDNF/TrkB/Akt signaling associated with synaptic plasticity. We found that Girdin in the hippocampus was phosphorylated at S1416 in an activity-dependent manner. Phosphorylation-deficient knock-in mice (GirdinSA/SAmice), in which S1416 is replaced with alanine, exhibited shrinkage of spines, deficit of hippocampal long-term potentiation, and memory impairment. These phenotypes ofGirdinSA/SAmice resembled those ofGirdin+/−mice, which have 50% loss of Girdin expression. Furthermore, Girdin interacted with Src kinase and NR2B subunit of NMDA receptor, leading to phosphorylation of the NR2B subunit and NMDA receptor activation. Our findings suggest that Girdin has two different functions in the hippocampus: Akt-independent neuronal migration and Akt-dependent NR2B phosphorylation through the interaction with Src, which is associated with synaptic plasticity in the hippocampus underlying memory formation.

Although opioid peptides are involved in the regulation of the hypothalamic-pituitary-adrenal axis, their role in pro-opiomelanocortin (POMC) gene expression at the pituitary level is not known. We therefore examined the effects of opioid receptor agonists, including recently discovered endogenous opioid peptides, on POMC gene expression using the AtT20PL cell line, a subclone of AtT20 in which the rat POMC 5′-promoter-luciferase fusion gene was stably incorporated. The endogenous µ-opioid receptor agonists endomorphin 1 and 2 had no effect on either basal or corticotropin-stimulating-hormone-induced POMC expression. This was also the case with the δ-agonist BUBUC, the ĸ-agonist U50488H and the orphan receptor agonist orphanin FQ. In contrast, the synthetic µ-agonist loperamide significantly inhibited basal and yet enhanced cAMP-induced POMC expression. The inhibitory effect of loperamide was mimicked by the calmodulin antagonist W7 and antagonized by the calcium channel blocker nifedipine, whereas neither the inhibitory nor the enhancing effect of loperamide was influenced by the opioid antagonist naloxone. These results suggest that the synthetic µ-agonist loperamide has a modulatory effect on the 5′-promoter activity of the POMC gene. This effect does not seem to be mediated through the classical µ-opioid receptor but rather in part through a calcium/calmodulin-related mechanism.

CD109, a glycosylphosphatidylinositol-anchored glycoprotein, is expressed at high levels in some human tumors including squamous cell carcinomas. As CD109 is reportedly cleaved by furin and its soluble form is secreted into culture medium in vitro, we hypothesized that CD109 could serve as a tumor marker in vivo. In this study, we investigated CD109 as a novel serum tumor marker using transgenic mice that overexpress mouse CD109 (mCD109-TG mice) and tumor xenografted mice inoculated with human CD109 (hCD109)-overexpressing HEK293 cells. In sera and urine of mCD109-TG mice, mCD109 was detected using western blotting. In xenografted mice, hCD109 secreted from inoculated tumors was detected in sera, using western blotting and CD109 ELISA. Concentrations of tumor-secreted CD109 increased proportionally as tumors enlarged. Concentrations of secreted CD109 decreased notably by 17 h after tumor resection, and became undetectable 48 h after resection. The half-life of tumor-secreted CD109 was about 5.86±0.17 h. These results indicate that CD109 is present in serum as a soluble form, and suggest its potential as a novel tumor marker in patients with cancers that express CD109.

SummaryFor collective invasion, cancer cells form cohesive groups comprised of leading cells (LCs) at the forefront and following cells (FCs) at the rear. However, the molecular mechanisms that define LCs and FCs remain elusive. Here, we demonstrated that LCs, but not FCs, upregulated the expression of integrin β1 after the loss of intercellular adhesion. The LC-specific expression of integrin β1 was posttranscriptionally regulated by the TRIM27/MRTF-B complex in response to the loss of intercellular adhesion, thereby regulating the stability and translation of integrin β1 mRNA via microRNA-124 in LCs. Accordingly, depletion of TRIM27 and MRTF-B abrogated the upregulation of integrin β1 in LCs and blocked the invasion of cancer cell groups in vitro and in vivo. Therefore, our findings revealed that the specific function of LCs was defined by intrinsic mechanisms related to the presence of the cell’s free surface, providing insights into the regulation of intratumor heterogeneity.

We examined the role of intracellular calcium release in the regulation of CRH-induced ACTH secretion using the AtT20 corticotroph cell line. We found that ruthenium red, an inhibitor of ryanodine receptor, substantially diminished the secretory response, whereas Xestospongin C, an inositol 1,4,5-triphosphate receptor antagonist, had no effect. Expression of two ryanodine receptor subtypes (RyR1 and RyR3) was confirmed by RT-PCR. We also found that caffeine, a ryanodine receptor agonist, significantly stimulated, whereas thapsigargin, which causes depletion of intracellular calcium store, markedly diminished, the ACTH release. These results suggest that ryanodine receptor-mediated calcium-induced calcium release is involved in the regulation of CRH-induced ACTH release.

Girdin is an Akt substrate and actin-binding protein. Mice with germ-line deletions of Girdin (a non-conditional knockout, (ncKO)) exhibit complete postnatal lethality accompanied by growth retardation and neuronal cell migration defects, which results in hypoplasia of the olfactory bulb and granule cell dispersion in the dentate gyrus. However, the physiological and molecular abnormalities in Girdin ncKO mice are not fully understood. In this study, we first defined the distribution of Girdin in neonates (P1) and adults (6months or older) using β-galactosidase activity in tissues from ncKO mice. The results indicate that Girdin is expressed throughout the nervous system (brain, spinal cord, enteric and autonomic nervous systems). In addition, β-galactosidase activity was detected in non-neural tissues, particularly in tissues with high tensile force, such as tendons, heart valves, and skeletal muscle. In order to identify the cellular population where the Girdin ncKO phenotype originates, newly generated Girdin flox mice were crossed with nestin promoter-driven Cre transgenic mice to obtain Girdin conditional knockout (cKO) mice. The phenotype of Girdin cKO mice was almost identical to ncKO mice, including postnatal lethality, growth retardation and decreased neuronal migration. Our findings indicate that loss of Girdin in the nestin cell lineage underlies the phenotype of Girdin ncKO mice.

Dishevelled is the common mediator of canonical and non-canonical Wnt signalling pathways, which are important for embryonic development, tissue maintenance and cancer progression. In the non-canonical Wnt signalling pathway, the Rho family of small GTPases acting downstream of Dishevelled has essential roles in cell migration. The mechanisms by which the non-canonical Wnt signalling pathway regulates Rac activation remain unknown. Here we show that Daple (Dishevelled-associating protein with a high frequency of leucine residues) regulates Wnt5a-mediated activation of Rac and formation of lamellipodia through interaction with Dishevelled. Daple increases the association of Dishevelled with an isoform of atypical protein kinase C, consequently promoting Rac activation. Accordingly, Daple deficiency impairs migration of fibroblasts and epithelial cells during wound healing in vivo. These findings indicate that Daple interacts with Dishevelled to direct the Dishevelled/protein kinase λ protein complex to activate Rac, which in turn mediates the non-canonical Wnt signalling pathway required for cell migration. The non-canonical Wnt signalling pathway mediates cell migration via the GTPase Rac but the detailed mechanism is unclear. In this study, the Daple protein is shown to regulate Wnt5a mediated activation of Rac, resulting in the formation of lamellipodia and cell migration.

Vascular endothelial growth factor (VEGF) is recognized as a principal mediator of vessel growth. VEGF regulates various endothelial cellular processes, including cell migration, proliferation, and survival, through the serine threonine protein kinase Akt. The Akt substrate girdin, an actin-binding protein, is known to regulate VEGF-mediated postnatal angiogenesis. However, the role of girdin and its phosphorylation in neonatal retinal vascular development and ocular pathological neovascularization in vivo has not been elucidated. In the present study, therefore, we investigated these processes using Girdin(+/-) mice lacking one copy of the girdin gene and girdin S1416A knockin (Girdin-KI(SA/SA)) mice in which the phosphorylation site of girdin is completely disrupted. We used three mouse models of pathological ocular neovascularization: oxygen-induced retinopathy (a mouse model of ischemic retinopathies), laser-induced choroidal neovascularization, and a human VEGF transgenic mouse that overexpresses human VEGF specifically in photoreceptor cells and generates pathological neovascularization in the retina. Neonatal vascular development was delayed and pathological neovascularization was decreased in both Girdin(+/-) mice and Girdin-KI(SA/SA) mice. These results demonstrate that girdin and its phosphorylation play an important role in neonatal vascular development and in pathological neovascularization in the retina.

CD109, a glycosylphosphatidylinositol-anchored glycoprotein, is highly expressed in several types of human cancer tissues, in particular, squamous cell carcinomas. In normal human tissues, human CD109 expression is limited to certain cell types including myoepithelial cells of the mammary, lacrimal, salivary, and bronchial glands and basal cells of the prostate and bronchial epithelium. Although CD109 has been reported to negatively regulate transforming growth factor-β signaling in keratinocytes in vitro , its physiologic role in vivo remains largely unknown. To investigate the function of CD109 in vivo , we generated CD109-deficient ( CD109 −/− ) mice. Although CD109 −/− mice were born normally, transient impairment of hair growth was observed. At histologic analysis, kinked hair shafts, ectatic hair follicles with an accumulation of sebum, and persistent hyperplasia of the epidermis and sebaceous glands were observed in CD109 −/− mice. Immunohistochemical analysis revealed thickening of the basal and suprabasal layers in the epidermis of CD109 −/− mice, which is where endogenous CD109 is expressed in wild-type mice. Although CD109 was reported to negatively regulate transforming growth factor-β signaling, no significant difference in levels of Smad2 phosphorylation was observed in the epidermis between wild-type and CD109 −/− mice. Instead, Stat3 phosphorylation levels were significantly elevated in the epidermis of CD109 −/− mice compared with wild-type mice. These results suggest that CD109 regulates differentiation of keratinocytes via a signaling pathway involving Stat3.

In postnatally developing and adult brains, interneurons of the olfactory bulb (OB) are continuously generated at the subventricular zone of the forebrain. The newborn neuroblasts migrate tangentially to the OB through a well defined pathway, the rostral migratory stream (RMS), where the neuroblasts undergo collective migration termed “chain migration.” The cell-intrinsic regulatory mechanism of neuroblast chain migration, however, has not been uncovered. Here we show that mice lacking the actin-binding Akt substrate Girdin (a protein that interacts with Disrupted-In-Schizophrenia 1 to regulate neurogenesis in the dentate gyrus) have profound defects in neuroblast chain migration along the RMS. Analysis of two gene knock-in mice harboring Girdin mutants identified unique amino acid residues in Girdin's C-terminal domain that are responsible for the regulation of neuroblast chain migration but revealed no apparent requirement of Girdin phosphorylation by Akt. Electron microscopic analyses demonstrated the involvement of Girdin in neuroblast cell–cell interactions. These findings suggest that Girdin is an important intrinsic factor that specifically governs neuroblast chain migration along the RMS.

Whether insulin or IGFs regulate glycogen synthesis in the fetal liver remains to be determined. In this study, we used several knockout mouse strains, including those lacking Pdx-1 (pancreatic duodenal homeobox-1), Insr (insulin receptor), and Igf2 (IGF-II) to determine the role of these genes in the regulation of fetal hepatic glycogen synthesis. Our data show that insulin deficiency does not alter hepatic glycogen stores, whereas Insr and Igf2 deficiency do. We found that both insulin receptor isoforms (IR-A and IR-B) are present in the fetal liver, and their expression is gestationally regulated. IR-B is highly expressed in the fetal liver; nonetheless, the percentage of hepatic IR-A isoform, which binds Igf2, was significantly higher in the fetus than the adult. In vitro experiments demonstrate that Igf2 increases phosphorylation of hepatic Insr, insulin receptor substrate-2, and Akt proteins and also the activity of glycogen synthase. Igf2 ultimately increased glycogen synthesis in fetal hepatocytes. This increase could be blocked by the phosphoinositide 3-kinase inhibitor LY294008. Taken together, we propose Igf2 as a major regulator of fetal hepatic glycogen metabolism, the insulin receptor as its target receptor, and phosphoinositide 3-kinase as the signaling pathway leading to glycogen formation in the fetal liver.

While lipopolysaccharides (LPS) are known to activate the hypothalamo-pituitary-adrenal axis, their direct effects on proopiomelanocortin (POMC) and adrenocorticotropin (ACTH) expression at the pituitary level through Toll-like receptors (TLRs) remain unclear. In this study, we examined the effects of LPS on ACTH secretion and the transcription of the POMC gene in the AtT20 mouse pituitary corticotroph cell line. RT-PCR analysis showed that TLR1-4 and 6 subtype mRNAs were expressed in AtT20 cells. When the cells were treated with LPS, a significant increase in the 5'-promoter activity of POMC gene was observed at 24 h, without any stimulatory effect on ACTH secretion. LPS also stimulated the expression of c-Fos gene and protein, and AP1-, but not NF-kappaB-, mediated transcription. Overall, our data show the expression of TLRs in the pituitary corticotroph cells, and suggest the direct stimulatory effect of LPS on POMC gene expression via TLR (probably TLR4), although the intracellular signaling pathways in the corticotroph may be different from those in immune cells.

Cushing's disease is characterized by persistent ACTH secretion under hypercortisolemia. In an attempt to clarify the molecular mechanism, we examined the effect of 11beta-hydroxysteroid dehydrogenase (HSD) inhibition on glucocorticoid suppression of ACTH release using murine corticotroph tumor cells. We found that 11beta-HSD2, as well as -HSD1, was expressed in the cells and that its inhibition by carbenoxolone significantly improved the negative feedback effect of glucocorticoid. Carbenoxolone also enhanced apoptosis induced by cortisol. These effects are most likely attributable to inhibition of 11beta-HSD2 because only cortisol, a substrate of 11beta-HSD2, was present in these experimental conditions. We conclude that ectopic expression of 11beta-HSD2 is, at least in part, responsible for the impaired glucocorticoid suppression in corticotroph adenoma. Inhibition of 11beta-HSD2 may be applicable to the medical therapy for Cushing's disease.

Up-regulation of hypothalamo-pituitary-adrenal axis is maintained during acute inflammation and/or infection, in the face of sustained elevation of plasma glucocorticoid hormone. Inflammatory stress is usually associated with high plasma cytokine levels and increased generation of reactive oxygen species (ROS) as well. In this study, we examined the effect of ROS on the negative feedback regulation of glucocorticoid in hypothalamo-pituitary-adrenal axis using AtT20 corticotroph cells in vitro. When the cells were treated with H2O2, glucocorticoid suppression on the proopiomelanocortin gene promoter activity was attenuated in a dose-dependent manner. H2O2 also inhibited the ligand-stimulated nuclear translocation of glucocorticoid receptor. The released glucocorticoid suppression by H2O2 was not observed when the cells were cotreated with antioxidants. Together, these results suggest that increased ROS generation in the oxidative redox state attenuates the glucocorticoid negative feedback system, at least in part, by interfering with the nuclear translocation of glucocorticoid receptor and eliminating the repression on proopiomelanocortin gene expression.