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.

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.

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.

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.

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.

Live attenuated smallpox vaccine candidate (KVAC103) efficiently induces protective immune responses in mice.

Smallpox, caused by the variola virus belonging to the genus Orthopoxvirus, is an acute contagious disease that killed 300 million people in the 20th century. Since it was declared to be eradicated and the national immunization program against it was stopped, the variola virus has become a prospective bio-weapon. It is necessary to develop a safe vaccine that protects people from terrorism using this biological weapon and that can be administered to immunocompromised people. Our previous study reported on the development of an attenuated smallpox vaccine (KVAC103). This study evaluated cellular and humoral immune responses to various doses, frequencies, and routes of administration of the KVAC103 strain, compared to CJ-50300 vaccine, and its protective ability against the wild-type vaccinia virus Western Reserve (VACV-WR) strain was evaluated. The binding and neutralizing-antibody titers increased in a concentration-dependent manner in the second inoculation, which increased the neutralizing-antibody titer compared to those after the single injection. In contrast, the T-cell immune response (interferon-gamma positive cells) increased after the second inoculation compared to that of CJ-50300 after the first inoculation. Neutralizing-antibody titers and antigen-specific IgG levels were comparable in all groups administered KVAC103 intramuscularly, subcutaneously, and intradermally. In a protective immunity test using the VACV-WR strain, all mice vaccinated with CJ-50300 or KVAC103 showed 100% survival. KVAC103 could be a potent smallpox vaccine that efficiently induces humoral and cellular immune responses to protect mice against the VACV-WR strain.

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.