Tip60/KAT5 Histone Acetyltransferase Is Required for Maintenance and Neurogenesis of Embryonic Neural Stem Cells.

Epigenetic regulation via epigenetic factors in collaboration with tissue-specific transcription factors is curtail for establishing functional organ systems during development. Brain development is tightly regulated by epigenetic factors, which are coordinately activated or inactivated during processes, and their dysregulation is linked to brain abnormalities and intellectual disability. However, the precise mechanism of epigenetic regulation in brain development and neurogenesis remains largely unknown. Here, we show that Tip60/KAT5 deletion in neural stem/progenitor cells (NSCs) in mice results in multiple abnormalities of brain development. Tip60-deficient embryonic brain led to microcephaly, and proliferating cells in the developing brain were reduced by Tip60 deficiency. In addition, neural differentiation and neuronal migration were severely affected in Tip60-deficient brains. Following neurogenesis in developing brains, gliogenesis started from the earlier stage of development in Tip60-deficient brains, indicating that Tip60 is involved in switching from neurogenesis to gliogenesis during brain development. It was also confirmed in vitro that poor neurosphere formation, proliferation defects, neural differentiation defects, and accelerated astrocytic differentiation in mutant NSCs are derived from Tip60-deficient embryonic brains. This study uncovers the critical role of Tip60 in brain development and NSC maintenance and function in vivo and in vitro.

Loss of mitochondrial transcription factor A in neural stem cells leads to immature brain development and triggers the activation of the integral stress response in vivo.

Mitochondrial dysfunction is significantly associated with neurological deficits and age-related neurological diseases. While mitochondria are dynamically regulated and properly maintained during neurogenesis, the manner in which mitochondrial activities are controlled and contribute to these processes is not fully understood. Mitochondrial transcription factor A (TFAM) contributes to mitochondrial function by maintaining mitochondrial DNA (mtDNA). To clarify how mitochondrial dysfunction affects neurogenesis, we induced mitochondrial dysfunction specifically in murine neural stem cells (NSCs) by inactivating Tfam. Tfam inactivation in NSCs resulted in mitochondrial dysfunction by reducing respiratory chain activities and causing a severe deficit in neural differentiation and maturation both in vivo and in vitro. Brain tissue from Tfam-deficient mice exhibited neuronal cell death primarily at layer V and microglia were activated prior to cell death. Cultured Tfam-deficient NSCs showed a reduction in reactive oxygen species produced by the mitochondria. Tfam inactivation during neurogenesis resulted in the accumulation of ATF4 and activation of target gene expression. Therefore, we propose that the integrated stress response (ISR) induced by mitochondrial dysfunction in neurogenesis is activated to protect the progression of neurodegenerative diseases.

Developmentally Regulated RNA-binding Protein 1 (Drb1)/RNA-binding Motif Protein 45 (RBM45), a Nuclear-Cytoplasmic Trafficking Protein, Forms TAR DNA-binding Protein 43 (TDP-43)-mediated Cytoplasmic Aggregates.

Cytoplasmic protein aggregates are one of the pathological hallmarks of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Several RNA-binding proteins have been identified as components of inclusion bodies. Developmentally regulated RNA-binding protein 1 (Drb1)/RNA-binding motif protein 45 is an RNA-binding protein that was recently described as a component in ALS- and FTLD-related inclusion bodies. However, the molecular mechanism underlying cytoplasmic Drb1 aggregation remains unclear. Here, using an in vitro cellular model, we demonstrated that Drb1 co-localizes with cytoplasmic aggregates mediated by TAR DNA-binding protein 43, a major component of ALS and FTLD-related inclusion bodies. We also defined the domains involved in the subcellular localization of Drb1 to clarify the role of Drb1 in the formation of cytoplasmic aggregates in ALS and FTLD. Drb1 predominantly localized in the nucleus via a classical nuclear localization signal in its carboxyl terminus and is a shuttling protein between the nucleus and cytoplasm. Furthermore, we identify a double leucine motif serving as a nuclear export signal. The Drb1 mutant, presenting mutations in both nuclear localization signal and nuclear export signal, is prone to aggregate in the cytoplasm. The mutant Drb1-induced cytoplasmic aggregates not only recruit TAR DNA-binding protein 43 but also decrease the mitochondrial membrane potential. Taken together, these results indicate that perturbation of Drb1 nuclear-cytoplasmic trafficking induces toxic cytoplasmic aggregates, suggesting that mislocalization of Drb1 is involved in the cause of cytotoxicity in neuronal cells.

Rapid selection of XO embryonic stem cells using Y chromosome-linked GFP transgenic mice.

We developed a transgenic mouse line with Y chromosome-linked green fluorescent protein expressing transgenes (Y-GFP) by the conventional microinjection into the pronucleus of C57BL/6J fertilized oocytes. Embryonic stem (ES) cells derived from Y-GFP mice enabled not only sexing but also the identification of 39, XO karyotype by the lack of Y chromosome. Actually, when fluorescence activated cell sorting (FACS) was applied to Y-GFP ES cells, non-fluorescent ES cells were conveniently collected and showed the lack of Y chromosome by PCR genotyping and Southern blot analysis. FACS analysis revealed Y chromosome loss occurred at 2.9 % of 40, XY ES cells after five passages. These Y-GFP ES cells are potentially applicable to reduce the time, cost and effort needed to generate the gene-targeted mice by the production of male and female mice derived from the same ES cell clone.

Paraneoplastic cerebellar degeneration associated with an onconeural antibody against creatine kinase, brain-type.

Onconeural immunity, a cancer-stimulated immune reaction that cross-reacts with neural tissues, is considered to be the principal pathological mechanism for paraneoplastic neurological syndromes (PNS). A common PNS is paraneoplastic cerebellar degeneration (PCD). We had encountered a PCD patient with urothelial carcinomas (UC) of the urinary bladder who was negative for the well-characterized PNS-related onconeural antibodies. In the present study, we aimed to identify a new PCD-related onconeural antibody, capable of recognizing both cerebellar neurons and cancer tissues from the patient, and applied a proteomic approach using mass spectrometry. We identified anti-creatine kinase, brain-type (CKB) antibody as a new autoantibody in the serum and cerebrospinal fluid from the patient. Immunohistochemistry indicated that anti-CKB antibody reacted with both cerebellar neurons and UC of the urinary bladder tissues. However, anti-CKB antibody was not detected in sera from over 30 donors, including bladder cancer patients without PCD, indicating that anti-CKB antibody is required for onset of PCD. We also detected anti-CKB antibody in sera from three other PCD patients. Our study demonstrated that anti-CKB antibody may be added to the list of PCD-related autoantibodies and may be useful for diagnosis of PCD.

Translational repression of the McKusick-Kaufman syndrome transcript by unique upstream open reading frames encoding mitochondrial proteins with alternative polyadenylation sites.

BACKGROUND: Upstream open reading frames (uORFs) are commonly found in the 5'-untranslated region (UTR) of many genes and function in translational control. However, little is known about the existence of the proteins encoded by uORFs, and the role of the proteins except translational control. There was no report about uORFs of the McKusick-Kaufman syndrome (MKKS) gene that causes a genetic disorder. METHODS: Northern blotting, 3'-RACE, and bioinformatics were used for determining the length of transcripts and their 3' ends. Luciferase assay and in vitro translation were used for evaluation of translational regulatory activity of uORFs. Immunoblotting and immunocytochemical analyses were used for detection of uORF-derived protein products and their subcellular localization. RESULTS: The MKKS gene generates two types of transcripts: a canonical long transcript that encodes both uORFs and MKKS, and a short transcript that encodes only uORFs by using alternative polyadenylation sites at the 5'-UTR. The simultaneous disruption of the uORF initiation codons increased the translation of the downstream ORF. Furthermore, both protein products from the two longest uORFs were detected in the mitochondrial membrane fraction of HeLa cells. Database searches indicated that such uORFs with active alternative polyadenylation sites at the 5'-UTR are atypical but surely exist in human transcripts. CONCLUSIONS: Multiple uORFs at the 5'-UTR of the MKKS long transcript function as translational repressor for MKKS. Two uORFs are translated in vivo and imported onto the mitochondrial membrane. GENERAL SIGNIFICANCE: Our findings provide unique insights into production of uORF-derived peptides and functions of uORFs.

Epitope determination of anti rat thy-1 monoclonal antibody that regulates neurite outgrowth.

Glycosylphosphatidylinositol-anchored protein Thy-1 is abundantly expressed on the cell surface of neurons and T lymphocytes in rodents. Although Thy-1 is known to bind integrins as a ligand and to mediate neurite outgrowth and immune responses, its precise function is not fully understood. Previously we produced several anti rat Thy-1 monoclonal antibodies and identified one, 2E11, which induces PC12 cell neurite outgrowth. Here we screened antibodies that inhibit 2E11-induced neurite outgrowth and stimulate or inhibit rat thymocyte aggregation. Since Thy-1 lacks an intracellular region, it requires other membrane-bound molecules for the signal transduction. Hence these antibodies are hypothesized to play key roles in the interaction between Thy-1 and signaling molecules. To elucidate the mechanisms of antibody-induced Thy-1 functions, antibody characterization and epitope determination were carried out. Thy-1 cleavage and mutation revealed that the antibodies recognize not only amino acid sequences, but also the three-dimensional structures consisting of immunoglobulin-like domains. Two antibodies were suggested to bind spatially close to the integrin binding site and crosslink Thy-1 molecules, while a third antibody is believed to inhibit Thy-1 crosslinking and subsequent Thy-1 signaling. The antibodies reported here may therefore function as crosslinkers, agonists, or antagonists that modify Thy-1 signaling.

Characterization of ST2 transgenic mice with resistance to IL-33.

IL-33, a member of the IL-1 family, activates MAPK and NF-kappaB through its receptor ST2L and IL-1RAcP. ST2, a member of the IL-1R superfamily, is a secreted form of ST2 gene products, which has been shown to act as a decoy receptor for IL-33 and to inhibit the IL-33/ST2L/IL-1RAcP signaling pathway. In this work, we generated ST2 transgenic mice. In control mice, intraperitoneal administration of IL-33 caused an increased number of eosinophils in blood and in peritoneal cavity, an increased number of peritoneal M Phi, splenomegaly, accumulation of periodic acid-Schiff-positive material in the lung, and high concentrations of serum IL-5 and IL-13. However, these alterations were hardly detectable in ST2 Tg mice. In peritoneal M Phi from IL-33-stimulated mice, mRNA expression of M2 M Phi marker genes were increased compared with thioglycollate-elicited peritoneal M Phi. The IL-33-stimulation also increased the secretion of IL-6 from M Phi. However, when the IL-33 was preincubated with ST2 prior to its addition to the M Phi cultures, the secretion of IL-6 was attenuated. These data suggest that, though IL-33 induced the Th2-type immune responses and infiltration of M2 type M Phi into the peritoneal cavity, ST2 can downregulate these reactions both in vivo and in vitro.

Prostaglandin EP3 receptor superactivates adenylyl cyclase via the Gq/PLC/Ca2+ pathway in a lipid raft-dependent manner.

We previously demonstrated that prostaglandin EP3 receptor augments EP2-elicited cAMP formation in COS-7 cells in a G(i/o)-insensitive manner. The purpose of our current study was to identify the signaling pathways involved in EP3-induced augmentation of receptor-stimulated cAMP formation. The enhancing effect of EP3 receptor was irrespective of the C-terminal structure of the EP3 isoform. This EP3 action was abolished by treatment with inhibitors for phospholipase C and intracellular Ca(2+)-related signaling molecules such as U73122, staurosporine, 2-APB and SK&F 96365. Indeed, an EP3 agonist stimulated IP(3) formation and intracellular Ca(2+) mobilization, which was blocked by U73122, but not by pertussis toxin. The enhancing effect by EP3 on cAMP formation was mimicked by both a Ca(2+) ionophore and the activation of a typical G(q)-coupled receptor. Moreover, EP3 was exclusively localized to the raft fraction in COS-7 cells and EP3-elicited augmentation of cAMP formation was abolished by cholesterol depletion and introduction of a dominant negative caveolin-1 mutant. These results suggest that EP3 elicits adenylyl cyclase superactivation via G(q)/phospholipase C activation and intracellular Ca(2+) mobilization in a lipid raft microdomain-dependent manner.

Prostaglandin E2-EP4 signaling promotes immune inflammation through Th1 cell differentiation and Th17 cell expansion.

Two distinct helper T (TH) subsets, TH1 and TH17, mediate tissue damage and inflammation in animal models of various immune diseases such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases and allergic skin disorders. These experimental findings, and the implication of these TH subsets in human diseases, suggest the need for pharmacological measures to manipulate these TH subsets. Here we show that prostaglandin E2 (PGE2) acting on its receptor EP4 on T cells and dendritic cells not only facilitates TH1 cell differentiation but also amplifies interleukin-23-mediated TH17 cell expansion in vitro. Administration of an EP4-selective antagonist in vivo decreases accumulation of both TH1 and TH17 cells in regional lymph nodes and suppresses the disease progression in mice subjected to experimental autoimmune encephalomyelitis or contact hypersensitivity. Thus, PGE2-EP4 signaling promotes immune inflammation through TH1 differentiation and TH17 expansion, and EP4 antagonism may be therapeutically useful for various immune diseases.

Mechanism of destruction of microtubule structures by 4-hydroxy-2-nonenal.

A major end product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE), is an electrophilic alkenal and produces Michael adducts with cellular proteins. It is known that exposure of cultured cells to HNE causes rapid disappearance of microtubule networks. In this study we addressed the mechanism. Immunochemical studies revealed that HNE preferentially modified alpha-tubulin in rat primary neuronal cells, PC12 cells, and rat fibroblast cell line 3Y1 cells. This was morphologically associated with the disappearance of microtubule structures in those cells. In a purified rat brain microtubule fraction, HNE modified unpolymerized tubulin and impaired its polymerizability, with a concomitant increase in insolubilized tubulin. Nevertheless, HNE had a marginal effect on the stability of pre-polymerized microtubules. These results suggest that disruption of microtubule assembly as a result of HNE modification of unpolymerized tubulin, rather than destruction of assembled microtubules, is responsible for the disappearance of microtubule structures in cells exposed to HNE.

Structure-activity relationships of neoechinulin A analogues with cytoprotection against peroxynitrite-induced PC12 cell death.

Neoechinulin A, an alkaloid from Eurotium rubrum Hiji025, protected neuronal PC12 cells against cell death induced by peroxynitrite derived from SIN-1 (3-(4-morpholinyl)sydnonimine hydrochloride). In this study, we investigated the structure-activity relationships of neoechinulin A and a set of its analogues by using assays to measure anti-nitration and antioxidant activities and cytoprotection against SIN-1-induced PC12 cell death. The presence of the diketopiperazine ring was essential for both the antioxidant and anti-nitration activities of neoechinulin A derivatives. Nevertheless, a derivative lacking the diketopiperazine ring could still protect PC12 cells against SIN-1 cytotoxicity. An acyclic analogue completely lost the cytoprotective effect while retaining its antioxidant/anti-nitration activities. Pre-incubation of the cells with neoechinulin A for at least 12 hours was essential for the cells to gain SIN-1 resistance. These results suggest that neoechinulin A endows the cells with cytoprotection through a biological effect different from the apparent antioxidant/anti-nitration activities.

Molecular cloning of the chicken ST2 gene and a novel variant form of the ST2 gene product, ST2LV.

The ST2 gene encodes receptor-like molecules that are very similar to the interleukin-1 receptors. Three types of ST2 gene products, ST2, ST2L, and ST2V, can be produced by alternative splicing. In the course of cloning the chicken ST2 and ST2L cDNAs, we identified a novel variant cDNA other than ST2, ST2L, or ST2V, and tentatively named it ST2LV. ST2LV was produced by alternative splicing that deleted the transmembrane domain of ST2L. The chicken ST2 gene consisted of 13 exons and had two promoters followed by noncoding exons 1a and 1b, like the ST2 genes of human, mouse, and rat. The chicken ST2 mRNA was detected by RT-PCR as early as embryonic day 5. After that, the chicken ST2 mRNA was expressed in all examined organs, including the brain, eye, heart, lung, and liver. The chicken ST2LV mRNA was detected from embryonic day 10. The chicken ST2LV cDNA was transiently expressed in COS-7 cells. A protein of 69 kDa was detected in the culture supernatant, and the size of the protein was reduced to 53 kDa by treatment with peptide-N-glycosidase F, which suggested that ST2LV is a new soluble secreted and N-glycosylated variant of the ST2 gene product.

Activated, but not resting human Th2 cells, in contrast to Th1 and T regulatory cells, produce soluble ST2 and express low levels of ST2L at the cell surface.

The T1/ST2 gene encodes, as a result of differential splicing, a cell surface protein (transmembrane form of T1/ST2, ST2L) and a soluble, secreted, protein (ST2). Here, we show that transcripts for both ST2L and ST2 are present in activated human Th2 clones, but not in Th1 and T regulatory clones. This activation-dependent expression of ST2L/ST2 transcripts was also found in short-term in vitro differentiated, activated CD4(+) Th2 cells. No expression of ST2L or ST2 mRNA was detected in any of the resting T cell subsets. Low cell surface expression of ST2L was detected on activated Th2 clones, and on freshly isolated non-IFN-gamma-producing CD4(+) peripheral blood T cells, activated with anti-CD3 and anti-CD28 mAb. Finally, ST2 could be detected in the culture supernatants of activated, but not resting, Th2 clones. Taken together, these results show that the T1/ST2 gene products are inducible proteins and that human Th2 cells, in addition to expressing ST2L at their cell surface, secrete ST2 following activation.

Interleukin-1beta expression in human gastric carcinoma with Epstein-Barr virus infection.

The KT tumor is a transplantable strain of a human Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC), established in severe combined immunodeficiency (SCID) mice, with which the cytokine expression of EBVaGC can be investigated without interference from the infiltrating lymphocytes. As a part of a high-density oligonucleotide array (GeneChip) analysis of EBVaGC, the interleukin-1beta (IL-1beta) gene was the only cytokine gene that showed markedly higher expression in the KT tumor cells than in two tumor strains of EBV-negative GC. The results were confirmed by Northern blotting, Western blotting, and enzyme-linked immunosorbent assay. Furthermore, we demonstrated a positive signal for IL-1beta mRNA in the carcinoma cells of a surgically resected EBVaGC, but not in EBV-negative GC, by in situ hybridization. In vitro, IL-1beta increased the cell growth of a GC cell line, TMK1. Thus, IL-1beta may act as an autocrine growth factor in EBVaGC.