The CRHR1/CREB/REST signaling cascade regulates mammalian embryonic neural stem cell properties.

Growing evidence suggests that the corticotropin-releasing hormone (CRH) signaling pathway, mainly known as a critical initiator of humoral stress responses, has a role in normal neuronal physiology. However, despite the evidence of CRH receptor (CRHR) expression in the embryonic ventricular zone, the exact functions of CRH signaling in embryonic brain development have not yet been fully determined. In this study, we show that CRHR1 is required for the maintenance of neural stem cell properties, as assessed by in vitro neurosphere assays and cell distribution in the embryonic cortical layers following in utero electroporation. Identifying the underlying molecular mechanisms of CRHR1 action, we find that CRHR1 functions are accomplished through the increasing expression of the master transcription factor REST. Furthermore, luciferase reporter and chromatin immunoprecipitation assays reveal that CRHR1-induced CREB activity is responsible for increased REST expression at the transcriptional level. Taken together, these findings indicate that the CRHR1/CREB/REST signaling cascade plays an important role downstream of CRH in the regulation of neural stem cells during embryonic brain development.

YAP inhibits HCMV replication by impairing STING-mediated nuclear transport of the viral genome.

YES-associated protein (YAP), a critical actor of the mammalian Hippo signaling pathway involved in diverse biological events, has gained increased recognition as a cellular factor regulated by viral infections, but very few studies have investigated their relationship vice versa. In this study, we show that YAP impairs HCMV replication as assessed by viral gene expression analysis and progeny assays, and that this inhibition occurs at the immediate-early stages of the viral life cycle, at the latest. Using YAP mutants lacking key functional domains and shRNA against TEAD, we show that the inhibitory effects of YAP on HCMV replication are nuclear localization- and TEAD cofactor-dependent. Quantitative real-time PCR (qPCR) and subcellular fractionation analyses reveal that YAP does not interfere with the viral entry process but inhibits transport of the HCMV genome into the nucleus. Most importantly, we show that the expression of stimulator of interferon genes (STING), recently identified as an important component for nuclear delivery of the herpesvirus genome, is severely downregulated by YAP at the level of gene transcription. The functional importance of STING is further confirmed by the observation that STING expression restores YAP-attenuated nuclear transport of the HCMV genome, viral gene expression, and progeny virus production. We also show that HCMV-upregulated YAP reduces expression of STING. Taken together, these findings indicate that YAP possesses both direct and indirect regulatory roles in HCMV replication at different infection stages.

Gamma secretase inhibition impairs HCMV replication by reduction of immediate early gene expression at the transcriptional level.

Due to diverse pathogenic potentials, there is a growing need for anti-HCMV agents. In this study, we show that treatment with DAPT, a γ-secretase inhibitor (GSI), impairs HCMV replication as assessed by a progeny assay based on immunostaining. This effect is not limited to DAPT because other GSIs with different structures and distinct mechanisms of action also exhibit a similar level of inhibitory effects on HCMV viral production, indicating that γ-secretase activity is required for efficient HCMV replication. Western blot and qPCR analyses reveal that DAPT does not interfere with the viral entry process, but reduces expression of the immediate early protein IE1 at the transcriptional level. Furthermore, we exclude the possible involvement of Notch signaling pathway during HCMV replication by showing that expression of the dominant-negative form of MAML1, which disrupts the transactivational ability of Notch intracellular domain (NICD), does not reduce viral particle formation, and that NICD cannot rescue the DAPT-treated outcomes. Taken together, these findings indicate that γ-secretase activity plays an important role in a key step of the HCMV life cycle and γ-secretase inhibition could potentially be used as a novel preventive and therapeutic strategy against HCMV infection and HCMV-related diseases.

YAP Enhances FGF2-Dependent Neural Stem Cell Proliferation by Induction of FGF Receptor Expression.

The Hippo signaling pathway regulates cell proliferation and organ growth, and its activation is mainly reflected by the phosphorylation levels of Yes-associated protein (YAP). In this study, we show that YAP facilitates embryonic neural stem cell proliferation by elevating their responsiveness to fibroblast growth factor 2 (FGF2), one of the major growth factors for neural stem cells, in vivo as well as in vitro. Western blot and quantitative real-time PCR analyses revealed that expression of the FGF receptors (FGFRs) FGFR1 to FGFR4 were greatly increased by YAP expression upon FGF2 treatment, followed by upregulation of the mitogen-activated protein kinase and protein kinase B signaling pathways. Furthermore, as assessed by quantitative real-time PCR analyses, YAP-induced FGFR expression was found to be TEA domain transcription factor (TEAD)-independent, and transcriptional coactivator with PDZ-binding motif, the other homolog of Yorki in the Drosophila Hippo signaling pathway, was found to possess similar activity to YAP. Finally, adjustment of FGFR signaling activity in the YAP-expressing cells to control levels efficiently offset the cell proliferative effects of YAP, suggesting that the increased proliferation of YAP-expressing neural stem cells was mainly attributable to enhanced FGFR signaling. Our data indicate that YAP plays an important role in neural stem cell regulation by elevating FGFR expression, subsequently leading to enhanced cell proliferation.

Non-cell autonomous promotion of astrogenesis at late embryonic stages by constitutive YAP activation.

Although astrocytes have gained increased recognition as an important regulator in normal brain function and pathology, the mechanisms underlying their genesis are not well understood. In this study, we show that constitutive YAP activation by in utero introduction of a non-degradable form of the YAP gene (YAP 5SA) causes productive GFAP+ cell generation at late embryonic periods, and this activity is nuclear localization- and TEAD transcription factor-dependent. Moreover, we found that the GFAP+ cells were not YAP 5SA-expressing cells themselves but cells in the vicinity in vivo. Conditioned medium prepared from YAP 5SA-expressing cells induced GFAP+ cell production in vitro, suggesting that a soluble factor(s) was mediating the astrogenic activity of YAP 5SA. Indeed, YAP 5SA expression greatly increased CNTF and BMP4 transcription in neural progenitor cells, and a neutralizing antibody against CNTF reduced the astrogenic effects of YAP 5SA-conditioned medium. Furthermore, the YAP 5SA-expressing cells were identified as FN1+ mesenchymal cells which are responsible for the precocious astrogenesis. These results suggest a novel molecular mechanism by which YAP activation can induce astrogenesis in a non-cell autonomous manner.

PACT increases mammalian embryonic neural stem cell properties by facilitating activation of the notch signaling pathway.

The protein activator of protein kinase R (PKR) (PACT) is known to play important roles in PKR regulation and microRNA biogenesis. Based on the observation that PACT is specifically expressed in the ventricular zone (VZ) at the mid-neurogenic period, we examine the role of PACT in this embryonic neural stem cell niche. Here, we provide the first evidence that PACT increases neurosphere formation, as well as expression of Notch target genes and the neural stem cell marker Sox2 in primary neural stem cells in vitro. Consistently, introduction of PACT into the mouse embryonic brain in utero increased the fraction of cells localizing to the VZ. We also show that the PACT-enhanced stemness of neural stem cells is PKR-independent. At the molecular level, PACT was revealed to physically interact with C promoter binding factor 1 (CBF1) and dramatically strengthen the association between CBF1 and Notch intracellular domain (NICD), which indicates stabilization of the Notch transcriptional coactivation complex responsible for Notch target gene expression. Taken together, our study indicates that PACT is a novel transcriptional coactivator of the Notch pathway playing a pivotal role during mammalian brain development.

Ttyh1 regulates embryonic neural stem cell properties by enhancing the Notch signaling pathway.

Despite growing evidence linking Drosophila melanogaster tweety-homologue 1 (Ttyh1) to normal mammalian brain development and cell proliferation, its exact role has not yet been determined. Here, we show that Ttyh1 is required for the maintenance of neural stem cell (NSC) properties as assessed by neurosphere formation and in vivo analyses of cell localization after in utero electroporation. We find that enhanced Ttyh1-dependent stemness of NSCs is caused by enhanced γ-secretase activity resulting in increased levels of Notch intracellular domain (NICD) production and activation of Notch targets. This is a unique function of Ttyh1 among all other Ttyh family members. Molecular analyses revealed that Ttyh1 binds to the regulator of γ-secretase activity Rer1 in the endoplasmic reticulum and thereby destabilizes Rer1 protein levels. This is the key step for Ttyh1-dependent enhancement of γ-secretase activity, as Rer1 overexpression completely abolishes the effects of Ttyh1 on NSC maintenance. Taken together, these findings indicate that Ttyh1 plays an important role during mammalian brain development by positively regulating the Notch signaling pathway through the downregulation of Rer1.

Human Cytomegalovirus IE2 Protein Disturbs Brain Development by the Dysregulation of Neural Stem Cell Maintenance and the Polarization of Migrating Neurons.

Despite the high incidence of severe defects in the central nervous system caused by human cytomegalovirus (HCMV) congenital infection, the mechanism of HCMV neuropathogenesis and the roles of individual viral genes have not yet been fully determined. In this study, we show that the immediate-early 2 (IE2) protein may play a key role in HCMV-caused neurodevelopmental disorders. IE2-transduced neural progenitor cells gave rise to neurospheres with a lower frequency and produced smaller neurospheres than control cells in vitro, indicating reduction of self-renewal and expansion of neural progenitors by IE2. At 2 days after in utero electroporation into the ventricle of the developing brain, a dramatically lower percentage of IE2-expressing cells was detected in the ventricular zone (VZ) and cortical plate (CP) compared to control cells, suggesting that IE2 concurrently dysregulates neural stem cell maintenance in the VZ and neuronal migration to the CP. In addition, most IE2+ cells in the lower intermediate zone either showed multipolar morphology with short neurites or possessed nonradially oriented processes, whereas control cells had long, radially oriented monopolar or bipolar neurites. IE2+ callosal axons also failed to cross the midline to form the corpus callosum. Furthermore, we provide molecular evidence that the cell cycle arrest and DNA binding activities of IE2 appear to be responsible for the increased neural stem cell exit from the VZ and cortical migrational defects, respectively. Collectively, our results demonstrate that IE2 disrupts the orderly process of brain development in a stepwise manner to further our understanding of neurodevelopmental HCMV pathogenesis.IMPORTANCE HCMV brain pathogenesis has been studied in limited experimental settings, such as in vitro HCMV infection of neural progenitor cells or in vivo murine CMV infection of the mouse brain. Here, we show that IE2 is a pivotal factor that contributes to HCMV-induced abnormalities in the context of the embryonic brain using an in utero gene transfer tool. Surprisingly, IE2, but not HCMV IE1 or murine CMV ie3, interferes pleiotropically with key neurodevelopmental processes, including neural stem cell regulation, proper positioning of migrating neurons, and the callosal axon projections important for communication between the hemispheres. Our data suggest that the wide spectrum of clinical outcomes, ranging from mental retardation to microcephaly, caused by congenital HCMV infection can be sufficiently explained in terms of IE2 action alone.

Neprilysin facilitates adipogenesis through potentiation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway.

Neprilysin (NEP) is a zinc metallopeptidase that cleaves a number of small peptides into inactive forms. Despite the recent evidence of a significant correlation between the levels of NEP in plasma and the severity of obesity in humans, a cause-and-effect relationship or a functional role of NEP in obesity has remained uncertain. In this study, we show that NEP has a positive regulatory effect on fat cell formation from precursor cells. NEP increases the accumulation of cytoplasmic triglycerides in 3T3-L1 preadipocytes or the C3H10T1/2 mesenchymal stem cell line in differentiation conditions. Consistently, cells expressing NEP showed an increase in mRNA expression of adipogenic transcription factors, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), and the adipocyte markers aP2 and adipsin. Furthermore, this NEP-enhanced induction of adipogenesis was found to require the enzymatic activity of NEP, leading to augmentation of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway. In summary, our results indicate that NEP accelerates adipogenesis through enhancement of insulin-mediated PI3K-Akt activation and imply a high therapeutic value of NEP in treating obesity and obesity-related disorders.

TRBP maintains mammalian embryonic neural stem cell properties by acting as a novel transcriptional coactivator of the Notch signaling pathway.

Transactivation response element RNA-binding protein (TRBP; TARBP2) is known to play important roles in human immunodeficiency virus (HIV) replication and microRNA biogenesis. However, recent studies implicate TRBP in a variety of biological processes as a mediator of cross-talk between signal transduction pathways. Here, we provide the first evidence that TRBP is required for efficient neurosphere formation and for the expression of neural stem cell markers and Notch target genes in primary neural progenitor cells in vitro Consistent with this, introduction of TRBP into the mouse embryonic brain in utero increased the fraction of cells expressing Sox2 in the ventricular zone. We also show that TRBP physically interacts with the Notch transcriptional coactivation complex through C promoter-binding factor 1 (CBF1; RBPJ) and strengthens the association between the Notch intracellular domain (NICD) and CBF1, resulting in increased NICD recruitment to the promoter region of a Notch target gene. Our data indicate that TRBP is a novel transcriptional coactivator of the Notch signaling pathway, playing an important role in neural stem cell regulation during mammalian brain development.

YAP/TAZ enhance mammalian embryonic neural stem cell characteristics in a Tead-dependent manner.

Mammalian brain development is regulated by multiple signaling pathways controlling cell proliferation, migration and differentiation. Here we show that YAP/TAZ enhance embryonic neural stem cell characteristics in a cell autonomous fashion using diverse experimental approaches. Introduction of retroviral vectors expressing YAP or TAZ into the mouse embryonic brain induced cell localization in the ventricular zone (VZ), which is the embryonic neural stem cell niche. This change in cell distribution in the cortical layer is due to the increased stemness of infected cells; YAP-expressing cells were colabeled with Sox2, a neural stem cell marker, and YAP/TAZ increased the frequency and size of neurospheres, indicating enhanced self-renewal- and proliferative ability of neural stem cells. These effects appear to be TEA domain family transcription factor (Tead)-dependent; a Tead binding-defective YAP mutant lost the ability to promote neural stem cell characteristics. Consistently, in utero gene transfer of a constitutively active form of Tead2 (Tead2-VP16) recapitulated all the features of YAP/TAZ overexpression, and dominant negative Tead2-EnR resulted in marked cell exit from the VZ toward outer cortical layers. Taken together, these results indicate that the Tead-dependent YAP/TAZ signaling pathway plays important roles in neural stem cell maintenance by enhancing stemness of neural stem cells during mammalian brain development.

Creatine inhibits adipogenesis by downregulating insulin-induced activation of the phosphatidylinositol 3-kinase signaling pathway.

Creatine is a nitrogenous organic acid known to function in adenosine triphosphate (ATP) metabolism. Recent evidence indicates that creatine regulates the differentiation of mesenchymal stem cells (MSCs) in processes such as osteogenesis and myogenesis. In this study, we show that creatine also has a negative regulatory effect on fat cell formation. Creatine inhibits the accumulation of cytoplasmic triglycerides in a dose-dependent manner irrespective of the adipogenic cell models used, including a C3H10T1/2 MSC line, 3T3-L1 preadipocytes, and primary human MSCs. Consistently, a dramatic reduction in mRNA expression of adipogenic transcription factors, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), glucose transporters, 1 and 4 (Glut1, Glut4), and adipocyte markers, aP2 and adipsin, was observed in the presence of creatine. Creatine appears to exert its inhibitory effects on adipogenesis during early differentiation, but not late differentiation, or proliferation stages through inhibition of the PI3K-Akt-PPARγ signaling pathway. In an in vivo model, administration of creatine into mice resulted in body mass increase without fat accumulation. In summary, our results indicate that creatine downregulates adipogenesis through inhibition of phosphatidylinositol 3-kinase (PI3K) activation and imply the potent therapeutic value of creatine in treating obesity and obesity-related metabolic disorders.

Notch intracellular domain deficiency in nuclear localization activity retains the ability to enhance neural stem cell character and block neurogenesis in mammalian brain development.

Notch has a broad range of regulatory functions in many developmental processes, including hematopoiesis, neurogenesis, and angiogenesis. Notch has several key functional regions such as the RBP-Jκ/CBF1 association module (RAM) domain, nuclear localization signals (NLS), and ankyrin (ANK) repeats. However, previous reports assessing the level of importance of these domains in the Notch signaling pathway are controversial. In this study, we have assessed the level of contribution of each Notch domain to the regulation of mammalian neural stem cells in vivo as well as in vitro. Reporter assays and real-time polymerase chain reactions show that the ANK repeats and RAM domain are indispensable to the transactivation of Notch target genes, whereas a nuclear export signal (NES)-fused Notch intracellular domain (NICD) mutant defective in nuclear localization exerts a level of activity comparable to unmodified NICD. Transactivational ability appears to be tightly coupled to Notch functions during brain development. Unlike ANK repeats and RAM domain deletion mutants, NES-NICD recapitulates NICD features such as promotion of astrogenesis at the expense of neurogenesis in vitro and enhancement of neural stem cell character in vivo. Our data support the previous observation that intranuclear localization is not essential to the oncogenesis of Notch1 in certain types of cells and imply the importance of the noncanonical Notch signaling pathway in the regulation of mammalian neural stem cells.