NF-Y-dependent regulation of glutamate receptor 4 expression and cell survival in cells of the oligodendrocyte lineage.

Glutamate receptor subunit 4 (GluA4) is highly expressed by neural cells sensitive to excitotoxicity, and is the predominant subunit expressed by oligodendrocyte precursor cells (OPC) during a key period of vulnerability to hypoxic-ischemic injury. Therefore, transcriptional networks downstream of excitotoxic GluA4 activation represent a promising area for therapeutic intervention. In this work, we identify the CCAAT binding transcription factor NF-Yb as a novel transcriptional regulator of Gria4 (GluA4 gene), and a controller of excitotoxic death in the oligodendroglial lineage. We describe a novel regulatory region within Gria4 containing CCAAT sequences whose binding by NF-Yb is regulated by excitotoxicity. Excitotoxicity-induced alterations in NF-Yb binding are associated with changes in Gria4 transcription, while knockdown of NF-Yb alters the transcription of reporter constructs containing this regulatory region. Data from immortalized and primary OPC reveal that RNAi and pharmacological disruption of NF-Yb alter Gria4 transcription, with the latter inducing apoptosis and influencing a set of apoptotic genes similarly regulated during excitotoxicity. These data provide the first definition of a trans-acting mechanism regulating Gria4, and identify the NF-Y network as a potential source of pharmacological targets for promoting OPC survival.

A possible mechanism for low affinity of silkworm Na+/K+-ATPase for K.

The affinity for K+ of silkworm nerve Na+/K+-ATPase is markedly lower than that of mammalian Na+/K+-ATPase (Homareda 2010). In order to obtain clues on the molecular basis of the difference in K+ affinities, we cloned cDNAs of silkworm (Bombyx mori) nerve Na+/K+-ATPase α and ß subunits, and analyzed the deduced amino acid sequences. The molecular masses of the α and ß subunits were presumed to be 111.5 kDa with ten transmembrane segments and 37.7 kDa with a single transmembrane segment, respectively. The α subunit showed 75% identity and 93% homology with the pig Na+/K+-ATPase α1 subunit. On the other hand, the amino acid identity of the ß subunit with mammalian counterparts was as low as 30%. Cloned α and ß cDNAs were co-expressed in cultured silkworm ovary-derived cells, BM-N cells, which lack endogenous Na+/K+-ATPase. Na+/K+-ATPase expressed in the cultured cells showed a low affinity for K+ and a high affinity for Na+, characteristic of the silkworm nerve Na+/K+-ATPase. These results suggest that the ß subunit is responsible for the affinity for K+ of Na+/K+-ATPase.

Proteomic identification of differentially expressed genes during differentiation of cynomolgus monkey (Macaca fascicularis) embryonic stem cells to astrocyte progenitor cells in vitro.

Understanding astrocytogenesis is valuable for the treatment of nervous system disorders, as astrocytes provide structural, metabolic and defense support to neurons, and regulate neurons actively. However, there is limited information about the molecular events associated with the differentiation from primate ES cells to astrocytes. We therefore investigated the differentially expressed proteins in early astrocytogenesis, from cynomolgus monkey ES cells (CMK6 cell line) into astrocyte progenitor (AstP) cells via the formation of primitive neural stem spheres (Day 4), mature neural stem spheres (NSS), and neural stem (NS) cells in vitro, using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). We identified 66 differentially expressed proteins involved in these five differentiation stages. Together with the results of Western blotting, RT-PCR, and a search of metabolic pathways related to the identified proteins, these results indicated that collapsin response mediator protein 2 (CRMP2), its phosphorylated forms, and cellular retinoic acid binding protein 1 (CRABP1) were upregulated from ES cells to Day 4 and NSS cells, to which differentiation stages apoptosis-associated proteins such as caspases were possibly related; Phosphorylated CRMP2s were further upregulated but CRABP1 was downregulated from NSS cells to NS cells, during which differentiation stage considerable axon guidance proteins for development of growth cones, axon attraction, and repulsion were possibly readied; Nonphosphorylated CRMP2 was downregulated but CRABP1 was re-upregulated from NS cells to AstP cells, in which differentiation stage reorganization of actin cytoskeleton linked to focal adhesion was possibly accompanied. These results provide insight into the molecular basis of early astrocytogenesis in monkey.

Single-molecule tracking of myelin basic protein during oligodendrocyte differentiation.

This study aimed to expand our understanding of myelin basic protein (MBP), a key component of central nervous system myelin, by developing a protocol to track and quantifying individual MBP particles during oligodendrocyte (OL) differentiation. MBP particle directionality, confinement, and diffusion were tracked by rapid TIRF and HILO imaging of Dendra2 tagged MBP in three stages of mouse oligodendroglia: OL precursors, early myelinating OLs, and mature myelinating OLs. The directionality and confinement of MBP particles increased at each stage consistent with progressive transport toward, and recruitment into, emerging myelin structures. Unexpectedly, diffusion data presented a more complex pattern with subpopulations of the most diffusive particles disappearing at the transition between the precursor and early myelinating stage, before reemerging in the membrane sheets of mature OLs. This diversity of particle behaviors, which would be undetectable by conventional ensemble-averaged methods, are consistent with a multifunctional view of MBP involving roles in myelin expansion and compaction.

Proteomic identification of differentially expressed genes in neural stem cells and neurons differentiated from embryonic stem cells of cynomolgus monkey (Macaca fascicularis) in vitro.

Understanding neurogenesis is valuable for the treatment of nervous system disorders. However, there is currently limited information about the molecular events associated with the transition from primate ES cells to neural cells. We therefore sought to identify the proteins involved in neurogenesis, from Macaca fascicularis ES cells (CMK6 cell line) to neural stem (NS) cells to neurons using two-dimensional gel electrophoresis (2-DE), peptide mass fingerprinting (PMF), and liquid chromatography-tandem mass spectrometry (LC-MS-MS). During the differentiation of highly homogeneous ES cells to NS cells, we identified 17 proteins with increased expression, including fatty acid binding protein 7 (FABP7), collapsin response mediator protein 2 (CRMP2), and cellular retinoic acid binding protein 1 (CRABP1), and seven proteins with decreased expression. In the differentiation of NS cells to neurons, we identified three proteins with increased expression, including CRMP2, and 10 proteins with decreased expression. Of these proteins, FABP7 is a marker of NS cells, CRMP2 is involved in axon guidance, and CRABP1 is thought to regulate retinoic acid access to its nuclear receptors. Western blot analysis confirmed the upregulation of FABP7 and CRABP1 in NS cells, and the upregulation of CRMP2 in NS cells and neurons. RT-PCR results showed that CRMP2 and FABP7 mRNAs were also upregulated in NS cells, while CRABP1 mRNA was unchanged. These results provide insight into the molecular basis of monkey neural differentiation.

Heat shock response enhanced by cell culture treatment in mouse embryonic stem cell-derived proliferating neural stem cells.

Cells have a regulatory mechanism known as heat shock (HS) response, which induces the expression of HS genes and proteins in response to heat and other cellular stresses. Exposure to moderate HS results in beneficial effects, such as thermotolerance and promotes survival, whereas excessive HS causes cell death. The effect of HS on cells depends on both exogenous factors, including the temperature and duration of heat application, and endogenous factors, such as the degree of cell differentiation. Neural stem cells (NSCs) can self-renew and differentiate into neurons and glial cells, but the changes in the HS response of symmetrically proliferating NSCs in culture are unclear. We evaluated the HS response of homogeneous proliferating NSCs derived from mouse embryonic stem cells during the proliferative phase and its effect on survival and cell death in vitro. The number of adherent cells and the expression ratios of HS protein (Hsp)40 and Hsp70 genes after exposure to HS for 20 min at temperatures above 43°C significantly increased with the extension of the culture period before exposure to HS. In contrast, caspase activity was significantly decreased by extension of the culture period before exposure to HS and suppressed the decrease in cell viability. These results suggest that the culture period before HS remarkably affects the HS response, influencing the expression of HS genes and cell survival of proliferating NSCs in culture.

Generation of Multipotential NG2 Progenitors From Mouse Embryonic Stem Cell-Derived Neural Stem Cells.

Embryonic stem cells (ESC) have the potential to generate homogeneous immature cells like stem/progenitor cells, which appear to be difficult to isolate and expand from primary tissue samples. In this study, we developed a simple method to generate homogeneous immature oligodendrocyte (OL) lineage cells from mouse ESC-derived neural stem cell (NSC). NSC converted to NG2+/OLIG2+double positive progenitors (NOP) after culturing in serum-free media for a week. NOP expressed Prox1, but not Gpr17 gene, highlighting their immature phenotype. Interestingly, FACS analysis revealed that NOP expressed proteins for NG2, but not PDGFRɑ, distinguishing them from primary OL progenitor cells (OPC). Nevertheless, NOP expressed various OL lineage marker genes including Cspg4, Pdgfrα, Olig1/2, and Sox9/10, but not Plp1 genes, and, when cultured in OL differentiation conditions, initiated transcription of Gpr17 and Plp1 genes, and expression of PDGFRα proteins, implying that NOP converted into a matured OPC phenotype. Unexpectedly, NOP remained multipotential, being able to differentiate into neurons as well as astrocytes under appropriate conditions. Moreover, NOP-derived OPC myelinated axons with a lower efficiency when compared with primary OPC. Taken together, these data demonstrate that NOP are an intermediate progenitor cell distinguishable from both NSC and primary OPC. Based on this profile, NOP may be useful for modeling mechanisms influencing the earliest stages of oligogenesis, and exploring the cellular and molecular responses of the earliest OL progenitors to conditions that impair myelination in the developing nervous system.

Proteomic identification of differentially expressed genes in mouse neural stem cells and neurons differentiated from embryonic stem cells in vitro.

Embryonic stem (ES) cells are pluripotent stem cells and give rise to a variety of differentiated cell types including neurons. To study a molecular basis for differentiation from ES cells to neural cells, we searched for proteins involved in mouse neurogenesis from ES cells to neural stem (NS) cells and neurons by two-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting, using highly homogeneous cells differentiated from ES cells in vitro. We newly identified seven proteins with increased expression and one protein with decreased expression from ES cells to NS cells, and eight proteins with decreased expression from NS cells to neurons. Western blot analysis confirmed that a tumor-specific transplantation antigen, HS90B, decreased, and an extracellular matrix and membrane glycoprotein (such as laminin)-binding protein, galectin 1 (LEG1), increased in NS cells, and LEG1 and a cell adhesion receptor, laminin receptor (RSSA), decreased in neurons. The results of RT-PCR showed that mRNA of LEG1 was also up-regulated in NS cells and down-regulated in neurons, implying an important role of LEG1 in regulating the differentiation. The differentially expressed proteins identified here provide insight into the molecular basis of neurogenesis from ES cells to NS cells and neurons.

Ramelteon, a selective MT1/MT2 receptor agonist, suppresses the proliferation and invasiveness of endometrial cancer cells.

The incidence of endometrial cancer is increasing, making it the fifth most common cancer worldwide. To date, however, there is no standard therapy for patients with recurrent endometrial cancer. Melatonin, a hormone secreted by the pineal gland, has been shown to have anti-tumor effects in various tumor types. Although melatonin is available as a supplement, it has not been approved for cancer treatment. Ramelteon, a selective melatonin receptor type 1 and 2 (MT1/MT2) receptor agonist, has been approved to treat sleep disorders, suggesting that ramelteon may be effective in the treatment of endometrial cancer. To determine whether this agent may be effective in the treatment of endometrial cancer, this study investigated the ability of ramelteon to suppress the proliferation and invasiveness of HHUA cells, an estrogen receptor-positive endometrial cancer cell line. Ramelteon at 10-8 M maximally suppressed the proliferation of HHUA cells, reducing the percentage of Ki-67 positive proliferating cells. This effect was completely blocked by luzindole, a MT1/MT2 receptor antagonist. Furthermore, ramelteon inhibited HHUA cell invasion and reduced the expression of the MMP-2 and MMP-9 genes. These results suggested that ramelteon may be a candidate for the treatment of recurrent endometrial cancer, with activity similar to that of melatonin.

Astrocytogenesis of embryonic stem-cell-derived neural stem cells: Default differentiation.

Neural stem cells differentiate from embryonic stem cells via formation of neural stem spheres under free-floating conditions in astrocyte-conditioned medium. Subsequent culture of neural stem spheres on an adhesive substrate with fibroblast growth factor-2 promotes the migration of neural stem cells onto the substrate, resulting in an increase in the number of cells. These embryonic stem cell-derived neural stem cells can be differentiated almost exclusively into astrocytes by withdrawing fibroblast growth factor-2 from the medium without any additional instructions.

Pluripotent stem cell-derived neural stem cells: From basic research to applications.

Basic research on pluripotent stem cells is designed to enhance understanding of embryogenesis, whereas applied research is designed to develop novel therapies and prevent diseases. Attainment of these goals has been enhanced by the establishment of embryonic stem cell lines, the technological development of genomic reprogramming to generate induced-pluripotent stem cells, and improvements in vitro techniques to manipulate stem cells. This review summarizes the techniques required to generate neural cells from pluripotent stem cells. In particular, this review describes current research applications of a simple neural differentiation method, the neural stem sphere method, which we developed.

Effects of heat shock on survival, proliferation and differentiation of mouse neural stem cells.

Hyperthermia during pregnancy is a significant cause of reproductive problems ranging from abortion to congenital defects of the central nervous system (CNS), including neural tube defects and microcephaly. Neural stem cells (NSCs) can proliferate and differentiate into neurons and glia, playing a key role in the formation of the CNS. Here, we examined the effects of heat shock on homogeneous proliferating NSCs derived from mouse embryonic stem cells. After heat shock at 42 °C for 20 min, the proliferating NSCs continued to proliferate, although subtle changes were observed in gene expression and cell survival and proliferation. In contrast, heat shock at 43 °C caused a variety of responses: the up-regulation of genes encoding heat shock proteins (HSP), induction of apoptosis, temporal inhibition of cell proliferation and retardation of differentiation. Finally, effects of heat shock at 44 °C were severe, with almost all cells disappearing and the remaining cells losing the capacity to proliferate and differentiate. These temperature-dependent effects of heat shock on NSCs may be valuable in elucidating the mechanisms by which hyperthermia during pregnancy causes various reproductive problems.

Localization of Na(+)/K(+)-ATPase in silkworm brain: a possible mechanism for protection of Na(+)/K(+)-ATPase from Ca(2+).

In mammalian blood, the Na(+) concentration is higher than the K(+) concentration, whereas in hemolymph of lepidopterous insects, the K(+) concentration is higher than the Na(+) concentration. Na(+)/K(+)-ATPase regulates Na(+) and K(+) concentrations in mammalian blood. Therefore, the absence of Na(+)/K(+)-ATPase in lepidopterous insects might be expected. However, we have observed that Na(+)/K(+)-ATPase is abundant in nerve tissues of larvae of silkworm, a lepidopterous insect. Furthermore, we found that silkworm Na(+)/K(+)-ATPase was completely inhibited by 3 mM Ca(2+)in vitro (Homareda, 2010), although the Ca(2+) concentration is very high (30-50 mM) in the hemolymph of silkworm larvae. To investigate the reason why silkworm Na(+)/K(+)-ATPase is not inhibited by Ca(2+)in vivo, we observed the localization of Na(+)/K(+)-ATPase in nerve tissues using immunohistochemical techniques. Na(+)/K(+)-ATPase was distributed in the cortex and neuropile but not in the perineurium of the silkworm brain, while plasma membrane Ca(2+)-ATPase appeared to distribute in the perineurium as well as in the cortex and neuropile. These results support a possibility that neuronal Na(+)/K(+)-ATPase is protected from a high Ca(2+) concentration by the blood-brain barrier consisting of perineurial glial cells with plasma membrane Ca(2+)-ATPase.

Proliferation and differentiation of neural stem cells irradiated with X-rays in logarithmic growth phase.

Exposure of the fetal brain to ionizing radiation causes congenital brain abnormalities. Normal brain formation requires regionally and temporally appropriate proliferation and differentiation of neural stem cells (NSCs) into neurons and glia. Here, we investigated the effects of X-irradiation on proliferating homogenous NSCs prepared from mouse ES cells. Cells irradiated with X-rays at a dose of 1Gy maintained the capabilities for proliferation and differentiation but stopped proliferation temporarily. In contrast, the cells ceased proliferation following irradiation at a dose of >5Gy. These results suggest that irradiation of the fetal brain at relatively low doses may cause congenital brain abnormalities as with relatively high doses.

Uni-directional differentiation of mouse embryonic stem cells into neurons by the neural stem sphere method.

We previously showed that our neural stem sphere (NSS) method promotes the neuronal differentiation of mouse, monkey and human embryonic stem (ES) cells. Here we analyzed changes in expression of marker genes and proteins during neuronal differentiation. When cultured in astrocyte-conditioned medium (ACM) under free-floating conditions, colonies of ES cells formed floating cell spheres, which, within 4 days, gave rise to NSSs. In the spheres, the expression of ES cell marker genes was consistently down-regulated, while expression of an epiblast marker was transiently up-regulated, beginning on day 2, and the expression of neuroectoderm, neural stem cell and neuron markers was up-regulated, beginning on days 3, 4 and 6, respectively. The expression of the marker genes was consistent with that of marker proteins. The time course of expression of these markers in the spheres resembled that of neuronal differentiation from the inner cell mass (ICM) cells of blastula. In contrast, the expression of endoderm, mesoderm, epidermis, astrocyte and oligodendrocyte markers was low and not up-regulated during differentiation. Only a small number of apoptotic cells were present in the spheres. These results suggest that mouse ES cells uni-directionally differentiate into neurons via epiblast cells, neuroectodermal cells and neural stem cells.