坂下 英司

Objectives Compared to conventional disease-modifying antirheumatic drugs (DMARDs), biological DMARDs demonstrate superior efficacy but come with higher costs and increased infection risks. The ability to stop and resume biological DMARD treatment while maintaining remission would significantly alleviate these barriers and anxieties. The objective of this study was to identify biomarkers that can predict an imminent relapse, hopefully enabling the timely resumption of biological DMARDs before relapse occurs. Methods Forty patients with rheumatoid arthritis who had been in remission for more than 12 months were included in the study. The patients discontinued their biological DMARD treatment and were monitored monthly for the next 24 months. Out of the 40 patients, 14 (35%) remained in remission at the end of the 24-month period, while 26 (65%) experienced relapses at different time points. Among the relapse cases, 13 patients experienced early relapse within 6 months, and another 13 patients had late relapse between 6 months and 24 months. Seventy-three cytokines in the sera collected longitudinally from the 13 patients with late relapse were measured by multiplex immunoassay. Using cytokines at two time points, immediately after withdrawal and just before relapse, volcano plot and area under the receiver operating characteristic curves (AUC) were drawn to select cytokines that distinguished imminent relapse. Univariate and multivariate logistic regression analyses were used for the imminent relapse prediction model. Results IL-6, IL-29, MMP-3, and thymic stromal lymphopoietin (TSLP) were selected as potential biomarkers for imminent relapse prediction. All four cytokines were upregulated at imminent relapse time point. Univariate and multivariate logistic regression showed that a combination model with IL-6, MMP-3, and TSLP yielded an AUC of 0.828 as top predictors of imminent relapse. Conclusions This methodology allows for the prediction of imminent relapse while patients are in remission, potentially enabling the implementation of on- and off-treatments while maintaining remission. It also helps alleviate patient anxiety regarding the high cost and infection risks associated with biological DMARDs, which are the main obstacles to benefiting from their superb efficacy.

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.

Abstract Since the advent of biological disease modifying anti-rheumatic drugs (bDMARDs) in the treatment of rheumatoid arthritis (RA), most RA patients receiving such drugs have achieved remission at the expense of cost and infection risk. After bDMARDs are withdrawn, a substantial proportion of patients would have relapses even if they were in complete remission. In our previous report, relapse prediction could be made at the time of bDMARD withdrawal by measuring the serum levels of five cytokines. We report herein that, among 73 cytokines examined, serum levels of only interferon β (IFNβ) at the time of bDMARD withdrawal could predict early relapse (within 5 months) in patients who were categorized to relapse by the five cytokines in our previous report, with a cut-off value of 3.38 in log₂ and AUC of 0.833. High serum levels of IFNβ in the early-relapse group remained high until actual relapse occurred. Therefore, patients who relapse early might be biochemically different from those who relapse late or do not relapse at all. We recommend that patients who are predicted to relapse early continue bDMARDs even if they are in complete remission. This finding contributes to shared decision-making regarding how and when bDMARDs should be discontinued.

Biological disease modifying anti-rheumatic drugs (bDMARDs) show dramatic treatment efficacy in rheumatoid arthritis (RA). Long-term use of bDMARDs, however, has disadvantages such as high costs and infection risk. Therefore, a methodology is needed to predict any future RA relapse. Herein, we report a novel multi-biomarker combination which predicts relapse after bDMARDs-withdrawal in patients in remission. Forty patients with RA in remission for more than 12 months were enrolled. bDMARDs were withdrawn and they were followed monthly for the next 24 months. Fourteen patients (35%) of 40 in the cohort remained in remission at 24 months, whereas 26 (65%) relapsed at various time-points. Serum samples obtained longitudinally from patients in remission were assessed for the relapse-prediction biomarkers and index from 73 cytokines by the exploratory multivariate ROC analysis. The relapse-prediction index calculated from the 5 cytokines, IL-34, CCL1, IL-1β, IL-2 and IL-19, strongly discriminated between patients who relapsed and those who stayed in remission. These findings could contribute to clinical decision-making as to the timing of when to discontinue bDMARDs in RA treatment.

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.

Gliomas with Isocitrate dehydrogenase 1 (IDH1) mutation have alterations in several enzyme activities, resulting in various metabolic changes. The aim of this study was to determine a mechanism for the better prognosis of gliomas with IDH mutation by performing metabolomic analysis. To understand the metabolic state of human gliomas, we analyzed clinical samples obtained from surgical resection of glioma patients (grades II-IV) with or without the IDH1 mutation, and compared the results with U87 glioblastoma cells overexpressing IDH1 or IDH1R132H. In clinical samples of gliomas with IDH1 mutation, levels of D-2-hydroxyglutarate (D-2HG) were increased significantly compared with gliomas without IDH mutation. Gliomas with IDH mutation also showed decreased intermediates in the tricarboxylic acid cycle and pathways involved in the production of energy, amino acids, and nucleic acids. The marked difference in the metabolic profile in IDH mutant clinical glioma samples compared with that of mutant IDH expressing cells includes a decrease in β-oxidation due to acyl-carnitine and carnitine deficiencies. These metabolic changes may explain the lower cell division rate observed in IDH mutant gliomas and may provide a better prognosis in IDH mutant gliomas.

Cytoplasmic protein aggregates are one of the pathological hallmarks of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FT LD). Several RNA-binding proteins have been identified as components of inclusion bodies. Developmentally regulated RNA-binding protein 1 (Drbl)/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 Drbl aggregation remains unclear. Here, using an in vitro cellular model, we demonstrated that Drbl 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 Drbl to clarify the role of Drbl in the formation of cytoplasmic aggregates in ALS and FTLD. Drbl 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 Drbl mutant, presenting mutations in both nuclear localization signal and nuclear export signal, is prone to aggregate in the cytoplasm. The mutant Drb 1-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 Drbl nuclear cytoplasmic trafficking induces toxic cytoplasmic aggregates, suggesting that mislocalization of Drbl is involved in the cause of cytotoxicity in neuronal cells.

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. (C) 2013 Elsevier B.V. All rights reserved.

Pre-mRNA splicing factors are often redistributed to nucleoli in response to physiological conditions and cell stimuli. In telophase nuclei, serine-arginine rich (SR) proteins, which usually reside in nuclear speckles, localize transiently to active ribosomal DNA (rDNA) transcription sites called nucleolar organizing region-associated patches (NAPs). Here, we show that ultraviolet light and DNA damaging chemicals induce the redistribution of SR and SR-related proteins to areas around nucleolar fibrillar components in interphase nuclei that are similar to, but distinct from, NAPs, and these areas have been termed DNA damage-induced NAPs (d-NAPs). In vivo labeling of nascent RNA distinguished d-NAPs from NAPs in that d-NAPs were observed even after full rDNA transcriptional arrest as a result of DNA damage. Studies under a variety of conditions revealed that d-NAP formation requires both RNA polymerase II-dependent transcriptional arrest and nucleolar segregation, in particular, the disorganization of the granular nucleolar components. Despite the redistribution of SR proteins, splicing factor-enriched nuclear speckles were not disrupted because other nuclear speckle components, such as nuclear poly(A) RNA and the U5-116K protein, remained in DNA-damaged cells. These data suggest that the selective redistribution of splicing factors contributes to the regulation of specific genes via RNA metabolism. Finally, we demonstrate that a change in alternative splicing of apoptosis-related genes is coordinated with the occurrence of d-NAPs. Our results reveal a novel response to DNA damage that involves the dynamic redistribution of splicing factors to nucleoli.

Human RNPS1 was originally characterized as a pre-mRNA splicing activator in vitro and was shown to regulate alternative splicing in vivo. RNPS1 was also identified as a protein component of the splicing-dependent mRNP complex, or exon-exon junction complex (EJC), and a role for RNPS1 in postsplicing processes has been proposed. Here we demonstrate that RNPS1 incorporates into active spliceosomes, enhances the formation of the ATP-dependent A complex, and promotes the generation of both intermediate and final spliced products. RNPS1 is phosphorylated in vivo and interacts with the CK2 (casein kinase II) protein kinase. Serine 53 (Ser-53) of RNPS1 was identified as the major phosphorylation site for CK2 in vitro, and the same site is also phosphorylated in vivo. The phosphorylation status of Ser-53 significantly affects splicing activation in vitro, but it does not perturb the nuclear localization of RNPS1. In vivo experiments indicated that the phosphorylation of RNPS1 at Ser-53 influences the efficiencies of both splicing and translation. We propose that RNPS1 is a splicing regulator whose activator function is controlled in part by CK2 phosphorylation.

Human RNPS1 was originally purified and characterized as a pre-mRNA splicing activator, and its role in the postsplicing process has also been proposed recently. To search for factors that functionally interact with RNPS1, we performed a yeast two-hybrid screen with a human cDNA library. Four factors were identified: p54 (also called SRp54; a member of the SR protein family), human transformer 2beta (hTra2beta; an exonic splicing enhancer-binding protein), hLucA (a potential component of U1 snRNP), and pinin (also called DRS and MemA; a protein localized in nuclear speckles). The N-terminal region containing the serine-rich (S) domain, the central RNA recognition motif (RRM), and the C-terminal arginine/serine/proline-rich (RS/P) domain of RNPS1 interact with p54, pinin, and hTra2beta, respectively. Protein-protein binding between RNPS1 and these factors was verified in vitro and in vivo. Overexpression of RNPS1 in HeLa cells induced exon skipping in a model beta-globin pre-mRNA and a human tra-2beta pre-mRNA. Coexpression of RNPS1 with p54 cooperatively stimulated exon inclusion in an ATP synthase gamma-subunit pre-mRNA. The RS/P domain and RRM are necessary for the exon-skipping activity, whereas the S domain is important for the cooperative effect with p54. RNPS1 appears to be a versatile factor that regulates alternative splicing of a variety of pre-mRNAs.

Hu proteins are RNA-binding proteins that are the vertebrate homologs of Drosophila ELAV, and are implicated in stabilization or enhanced translation of specific mRNAs with AU-rich elements (AREs) in the 3'-untranslated region. Here, using the yeast two-hybrid system, we show that neuron-specific Hu proteins can interact with themselves. Immuno precipitation assays demonstrated that the interaction between Hu proteins occurs in mammalian cells and is strongly enhanced in the presence of cellular RNA. Furthermore, using in situ chemical crosslinking assays, we found that HuD, one of the neuron-specific Hu proteins, multimerizes in cells. The crosslinked HuD multimers retain specific RNA-binding ability and can interact with additional Hu proteins. Consistent with this biochemical property, HuD showed granular distribution in two neurogenic cell lines. These results suggest that the RNA-bound form of HuD multimerizes cooperatively to form a specific granular structure that may serve as a site of post-transcriptional regulation of ARE-containing mRNAs.

Neural RNA recognition motif (RRM)-type RNA-binding proteins play essential roles in neural development. To search for a new member of neural RRM-type RNA-binding protein, we screened rat cerebral expression library with polyclonal antibody against consensus RRM sequences. We have cloned and characterized a rat cDNA that belongs to RRM-type RNA-binding protein family, which we designate as drb1. Orthologs of drb1 exist in human and mouse. The predicted amino acid sequence reveals an open reading frame of 476 residues with a corresponding molecular mass of 53 kDa and consists of four RNA-binding domains. drb1 gene is specifically expressed in fetal (E12, E16) rat brain and gradually reduced during development. In situ hybridization demonstrated neuron-specific signals in fetal rat brain. RNA-binding assay indicated that human Drb1 protein possesses binding preference on poly(C)RNA. These results indicate that Drb1 is a new member of neural RNA-binding proteins, which expresses under spatio-temporal control. (C) 2002 Elsevier Science (USA). All rights reserved.

Mitochondrial ATP synthase gamma-subunit (F(1)gamma) pre-mRNA undergoes alternative splicing in a tissue- or cell type-specific manner. Exon 9 of F(1)gamma pre-mRNA is specifically excluded in heart and skeletal muscle tissues and in acid-stimulated human fibrosarcoma HT1080 cells, rhabdomyosarcoma KYM-1 cells, and mouse myoblast C2C12 cells. Recently, we found a purine-rich exonic splicing enhancer (ESE) element on exon 9 via transgenic mice bearing F(1)gamma mutant minigenes and demonstrated that this ESE functions ubiquitously with exception of muscle tissue (Ichida, M., Hakamata, Y., Hayakawa, M., Ueno E., Ikeda, U., Shimada, K., Hamamoto, T., Kagawa, Y., Endo, H. (2000) J. Biol. Chent. 275, 15992-16001). Here, we identified an exonic negative regulatory element responsible for muscle-specific exclusion of exon 9 using both in vitro and in vivo splicing systems. A supplementation assay with nuclear extracts from HeLa cells and acid-stimulated HT1080 cells was performed for an in vitro reaction of muscle-specific alternative splicing of F(1)gamma minigene and revealed that the splicing reaction between exons 8 and 9 was the key step for regulation of muscle-specific exon exclusion. Polypyrimidine tract in intron 8 requires ESE on exon 9 for constitutive splice site selection. Mutation analyses on the F(1)gammaEx8-9 minigene using a supplementation assay demonstrated that the muscle-specific negative regulatory element is positioned in the middle region of exon 9, immediately downstream from ESE. Detailed mutation analyses identified seven nucleotides (5'-AGUUCCA-3') as a negative regulatory element responsible for muscle-specific exon exclusion. This element was shown to cause exon skipping in in vivo splicing systems using acid-stimulated HT1080 cells after transient transfection of several mutant F(1)gammaEx8-9-10 minigenes. These results demonstrated that the 5'- AGUUCCA-3' immediately downstream from ESE is a muscle-specific exonic splicing silencer (MS-ESS) responsible for exclusion of exon 9 in vivo and in vitro.

HuC is one of the RNA binding proteins which are suggested to play important roles in neuronal differentiation and maintenance. We cloned and sequenced cDNAs encoding a mouse protein which is homologous to human HuC (hHuC). The longest cDNA encodes a 367 amino acid protein with three RNA recognition motifs (RRMs) and displays 96% identity to hHuC. Northern blot analysis showed that two different mRNAs, of 5.3 and 4.3 kb, for mouse HuC (mHuC) are expressed specifically in brain tissue. Comparison of cDNA sequences with the corresponding genomic sequence revealed that alternative 3' splice site selection generates two closely related mHuC isoforms. Iterative in vitro RNA selection and binding analyses showed that both HuC isoforms can bind with almost identical specificity to sequences similar to the AU-rich element (ARE), which is involved in the regulation of mRNA stability. Functional domain mapping using mHuC deletion mutants showed that the first RRM binds to ARE, that the second RRM has no RNA binding activity by itself, but facilitates ARE binding by the first RRM and that the third RRM has specific binding activity for the poly(A) sequence.

The Drosophila gene Sex-lethal (Sxl) controls the processes of sex determination and dosage compensation. A Drosophila subobscura genomic fragment containing all the exons and the late and early promoters in the Sri gene of D. melanogaster was isolated. Early Sxl expression in D, subobscura seems to be controlled at the transcriptional level, possibly by the X:A signal. In the region upstream of the early Sxl transcription initiation site are two conserved regions suggested to be involved in the early activation of Sxl. Late Sri expression in D. subobscura produces four transcripts in adult females and males. In males, the transcripts have an additional exon which contains three translational stop codons so that a truncated, presumably nonfunctional Sri protein is produced. The Sxl pre-mRNA of D. subobscura lacks the poly-U sequence presented at the polypirimidine tract of the 3' splice site of the male-specific exon present in D. melanogaster. Introns 2 and 3 contain the Sri-binding poly-U stretches, whose localization in intron 2 varies but in intron 3 is conserved. The Sri protein is fully conserved at the amino acid level in both species.

The Drosophila Sex-lethal (Sxl) contains two RNA-binding domains (RBDs) which belong to the RNA recognition motif (RRM) group. Sxl binds to a specific uridine-rich sequence which is believed to be the major cis-acting element for the splicing regulation of the transformer (tra) mRNA precursor. Here we show evidence supporting the previous suggestion that Sxl recognizes the sequence context downstream of the uridine-rich sequence. In addition, by means of UV-crosslinking assays with Sxl deletion constructs, we have demonstrated that Sxl RNA binding requires both of its RBDs for specificity and strength. Moreover, by the yeast two-hybrid analysis, we found that homodimeric interaction occurs between two Sxl molecules. Interestingly, the amino- and carboxy-terminal regions outside of the Sxl RBDs are dispensable for such dimerization, indicating that the protein-protein interaction is also mediated by RBDs. Coprecipitation experiments in vitro showed that the protein-protein interaction seems to be RNA-dependent but greatly enhanced by addition of the specific RNA containing the Sxl binding site, suggesting that the conformational change which is induced on binding to RNA may facilitate the interaction between Sxl molecules.

The Drosophila sex-lethal (Sxl) protein, a regulator of somatic sexual differentiation, is an RNA binding protein with two potential RNA recognition motifs (RRMs). It is thought to exert its function on splicing by binding to specific RNA sequences within Sxl and transformer (tra) pre-mRNAs. To examine the Sxl RNA binding specificity in detail, we performed in vitro selection and amplification of ligand RNAs from a random sequence pool on the basis of affinity with Sxl protein. After three cycles of selection and amplification, we cloned and sequenced 17 cDNAs corresponding to the RNAs selected in vitro. Sequencing showed that most of the RNAs selected contain polyuridine stretches surrounded by purine residues. In vitro binding analysis revealed that the sequences of the in vitro selected RNAs with relatively high affinity for Sxl show similarity to that of the Sxl- and tra-regulated acceptor regions, including the invariant AG sequence for splicing. These results suggest that Sxl recognizes and preferentially binds to a polyuridine stretch with a downstream AG sequence.