Takumi Takizawa

DNA Methylation Is a Critical Cell-Intrinsic Determinant of Astrocyte Differentiation in the Fetal Brain

Astrocyte differentiation, which occurs late in brain development, is largely dependent on the activation of a transcription factor, STAT3. We show that astrocytes, as judged by glial fibrillary acidic protein (GFAP) expression, never emerge from neuroepithelial cells on embryonic day (E) 11.5 even when STAT3 is activated, in contrast to E14.5 neuroepithelial cells. A CpG dinucleotide within a STAT3 binding element in the GFAP promoter is highly methylated in E11.5 neuroepithelial cells, but is demethylated in cells responsive to the STAT3 activation signal to express GFAP. This CpG methylation leads to inaccessibility of STAT3 to the binding element. We suggest that methylation of a cell type-specific gene promoter is a pivotal event in regulating lineage specification in the developing brain.

BMP2-mediated alteration in the developmental pathway of fetal mouse brain cells from neurogenesis to astrocytogenesis

We show that when telencephalic neural progenitors are briefly exposed to bone morphogenetic protein 2 (BMP2) in culture, their developmental fate is changed from neuronal cells to astrocytic cells. BMP2 significantly reduced the number of cells expressing microtubule-associated protein 2, a neuronal marker, and cells expressing nestin, a marker for undifferentiated neural precursors, but BMP2 increased the number of cells expressing S100-β, an astrocytic marker. In telencephalic neuroepithelial cells, BMP2 up-regulated the expression of negative helix–loop–helix (HLH) factors Id1, Id3, and Hes-5 (where Hes is homologue of hairy and Enhancer of Split) that inhibited the transcriptional activity of neurogenic HLH transcription factors Mash1 and neurogenin. Ectopic expression of either Id1 or Id3 (where Id is inhibitor of differentiation) inhibited neurogenesis of neuroepithelial cells, suggesting an important role for these HLH proteins in the BMP2-mediated changes in the neurogenic fate of these cells. Because gliogenesis in the brain and spinal cord, derived from implanted neural stem cells or induced by injury, is responsible for much of the failure of neuronal regeneration, this work may lead to a therapeutic strategy to minimize this problem.

Treatment of spinal cord injury by transplantation of fetal neural precursor cells engineered to express BMP inhibitor

Spontaneous recovery after spinal cord injury is limited. Transplantation of neural precursor cells (NPCs) into lesioned adult rat spinal cord results in only partial functional recovery, and most transplanted cells tend to differentiate predominantly into astrocytes. In order to improve functional recovery after transplantation, it is important that transplanted neural precursor cells appropriately differentiate into cell lineages required for spinal cord regeneration. In order to modulate the fate of transplanted cells, we advocate transplanting gene-modified neural precursor cells. We demonstrate that gene modification to inhibit bone morphogenetic protein (BMP) signaling by noggin expression promoted differentiation of neural precursor cells into neurons and oligodendrocytes, in addition to astrocytes after transplantation. Furthermore, functional recovery of the recipient mice with spinal cord injury was observed when noggin-expressing neural precursor cells were transplanted. These observations suggest that gene-modified neural precursor cells that express molecules involved in cell fate modulation could improve central nervous system (CNS) regeneration.

Fate alteration of neuroepithelial cells from neurogenesis to astrocytogenesis by bone morphogenetic proteins

Bone morphogenetic proteins (BMPs), a class of cytokines belonging to the transforming growth factor-beta superfamily, have been shown to play a wide variety of roles during development including those in the central nervous system. We here report that BMP2, BMP4 and BMP7 have an equivalent potential to inhibit neurogenesis and concomitantly induce astrocytogenesis of mouse fetal neuroepithelial cells. We further show that these BMPs activate a promoter of the gene for negative helix-loop-helix (HLH) factor, Id1, which is known to inhibit the function of such neurogenic transcription factors as Mash1 and neurogenin. These results suggest that BMP2, BMP4 and BMP7 alternate the fate of neuroepithelial cells from neuronal type to astrocytic one via a common mechanism involving negative HLH factor.

Signaling crosstalk underlying synergistic induction of astrocyte differentiation by BMPs and IL‐6 family of cytokines

We here show that bone morphogenetic protein (BMP) 7 acted in synergy with the distinct type of cytokines, leukemia inhibitory factor (LIF) and interleukin (IL) 6 that are in the IL‐6 family, to induce astrocyte differentiation from neuroepithelial cells as assessed by expression of glial fibrillary acidic protein (GFAP). In this synergistic action, transcription factors, Smads and STAT3 (for signal transducer and activator of transcription 3) activated by respective group of cytokines, as well as a transcriptional coactivator p300 were essential. Taken together with our previous finding that the synergistic astrocyte induction by BMP2 and LIF is attributed to the complex formation of Smads and STAT3 bridged by p300, it is conceivable that this complex formation is a mechanism utilized in common by two different types of cytokines belonging to the BMP and IL‐6 families in order to synergistically induce astrocyte differentiation.

Inhibition of BMP2-induced, TAK1 kinase-mediated neurite outgrowth by Smad6 and Smad7

Background BMP2 is known to play a wide variety of roles, including some in the development of the nervous system. This cytokine has been reported to induce neurite outgrowth in rat pheochromocytoma PC12 cells via the activation of a p38 MAP kinase, although its regulatory mechanism remains largely to be elucidated.

Role of GLI2 in the growth of human osteosarcoma

The Hedgehog pathway functions as an organizer in embryonic development. Aberrant activation of the Hedgehog pathway has been reported in various types of malignant tumours. The GLI2 transcription factor is a key mediator of Hedgehog pathway but its contribution to neoplasia is poorly understood. To establish the role of GLI2 in osteosarcoma, we examined its expression by real‐time PCR using biopsy tissues. To examine the function of GLI2, we evaluated the growth of osteosarcoma cells and their cell cycle after GLI2 knockdown. To study the effect of GLI2 activation, we examined mesenchymal stem cell growth and the cell cycle after forced expression of GLI2. We found that GLI2 was aberrantly over‐expressed in human osteosarcoma biopsy specimens. GLI2 knockdown by RNA interferences prevented osteosarcoma growth and anchorage‐independent growth. Knockdown of GLI2 promoted the arrest of osteosarcoma cells in G1 phase and was accompanied by reduced protein expression of the cell cycle accelerators cyclin D1, SKP2 and phosphorylated Rb. On the other hand, knockdown of GLI2 increased the expression of p21cip1. In addition, over‐expression of GLI2 promoted mesenchymal stem cell proliferation and accelerated their cell cycle progression. Finally, evaluation of mouse xenograft models showed that GLI2 knockdown inhibited the growth of osteosarcoma in nude mice. Our findings suggest that inhibition of GLI2 may represent an effective therapeutic approach for patients with osteosarcoma. Copyright

Identification of Allergen Fractions of Wheat Flour Responsible for Anaphylactic Reactions to Wheat Products in Infants and Young Children

Wheat is a food allergen which occasionally causes anaphylactic reactions exclusively in young children. There is very little knowledge of the clinical outcome in cases of food-related anaphylaxis and the differences in the allergenic protein components of food involved, comparing individuals who have suffered from an anaphylactic reaction with other individuals. The objectives of the present study were to examine the clinical features of 7 young children who had experienced anaphylactic reactions after ingesting wheat flour-containing products, and to analyze the allergens in wheat flour responsible for the anaphylactic symptoms. We measured the total IgE levels and the levels of IgE antibodies specific to wheat flour and performed IgE immunoblotting, comparing the sera from these children with sera from patients with atopic dermatitis. All sera from children who had experienced anaphylactic reactions were found to be positive for IgE specific to wheat. IgE immunoblotting revealed that 3 of these 7 children had sera showing reactivity to components of the salt-soluble protein fraction (16, 35–67 and 94 kD) and salt-insoluble protein-containing fraction (16, 38 and 70 kD) and 4 had no sera showing reactivity to components of the salt-soluble fraction. Patients with atopic dermatitis showed similar staining patterns. Various proteins in wheat flour could be allergens responsible for anaphylaxis and atopic dermatitis in infants or young children. Our findings suggest that these two clinically diverse allergic diseases do not necessarily represent responses to different allergenic proteins of wheat.

Foxa2 acts as a co-activator potentiating expression of the Nurr1-induced DA phenotype via epigenetic regulation

Understanding how dopamine (DA) phenotypes are acquired in midbrain DA (mDA) neuron development is important for bioassays and cell replacement therapy for mDA neuron-associated disorders. Here, we demonstrate a feed-forward mechanism of mDA neuron development involving Nurr1 and Foxa2. Nurr1 acts as a transcription factor for DA phenotype gene expression. However, Nurr1-mediated DA gene expression was inactivated by forming a protein complex with CoREST, and then recruiting histone deacetylase 1 (Hdac1), an enzyme catalyzing histone deacetylation, to DA gene promoters. Co-expression of Nurr1 and Foxa2 was established in mDA neuron precursor cells by a positive cross-regulatory loop. In the presence of Foxa2, the Nurr1-CoREST interaction was diminished (by competitive formation of the Nurr1-Foxa2 activator complex), and CoREST-Hdac1 proteins were less enriched in DA gene promoters. Consequently, histone 3 acetylation (H3Ac), which is responsible for open chromatin structures, was strikingly increased at DA phenotype gene promoters. These data establish the interplay of Nurr1 and Foxa2 as the crucial determinant for DA phenotype acquisition during mDA neuron development.

Chromatin Immunoprecipitation in Mouse Hippocampal Cells and Tissues

Chromatin immunoprecipitation (ChIP) has been developed for studying protein-DNA interactions and has been extensively used for mapping the localization of posttranslationally modified histones, histone variants, transcription factors, or chromatin modifying enzymes at a given locus or on a genome-wide scale. ChIP methods have been modified and improved over the years to fit a variety of different cell types and tissues. Here, we present a detailed protocol for hippocampal ChIP, of both minced tissue and enzyme-separated hippocampal cells. This protocol enables to study chromatin-protein interactions in a specified population of hippocampal cells, allowing to study chromatin regulation in the central nervous system in a variety of conditions and disorders. Our assay has been developed for histone modifications but is suited for any chromatin binding protein for which specific ChIP-grade antibodies are available.