Publications by authors named "Shu Aizawa"

21 Publications

  • Page 1 of 1

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Tyrosinase deficiency increases protein carbonyl content in substantia nigra of mice administered retinol palmitate.

Neuroreport 2021 01;32(2):121-124

Laboratory of Animal Genetics and Physiology, Department of Animal Science, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa, Japan.

Tyrosinase is a key enzyme for the biosynthesis of melanin pigments in peripheral tissues such as skin and retina. Although tyrosinase activity is specifically detected in melanocytes, several studies have shown the expression and enzymatic activity of tyrosinase in the central nervous system, especially in the midbrain substantia nigra. In the present study, we investigated the antioxidative effects of tyrosinase on protein damage in the substantia nigra of mice. C57BL/10JMsHir (B10) and tyrosinase-deficient albino B10.C-Tyrc/Hir (B10-c) mice were intraperitoneally administered retinol palmitate to induce oxidative stress, and the protein carbonyl content, a hallmark of protein oxidative damage, was examined in the substantia nigra. Retinol palmitate administration was found to decrease catalase activity in the substantia nigra of both B10 and B10-c mice, suggesting the induction of oxidative stress due to imbalanced antioxidant systems. In this model, we found that tyrosinase deficiency markedly increases the protein carbonyl content in the substantia nigra. Thus, we concluded that tyrosinase activity prevents protein damage in the substantia nigra of mice that were challenged with oxidative stress. These findings provide novel insight into the physiological role of tyrosinase in the central nervous system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/WNR.0000000000001566DOI Listing
January 2021

Cow milk exosomes activate NK cells and γδT cells in human PBMCs in vitro.

Immunol Med 2020 Dec 10;43(4):161-170. Epub 2020 Jul 10.

Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, Tokyo, Japan.

Cow milk is a nourishing food containing numerous essential nutrients. In Japan, the consumption of cow milk is thought to enhance resistance to exhaustion-related diseases. Although several nutrients in cow milk, such as lactoferrin, are thought to modulate immune cells, the mechanisms remain unclear. Recently, the immunoregulatory functions of food-derived microRNAs or exosomes have been reported. Therefore, we studied the effects of exosomes derived from cow milk (CM-Exs) on immune cells in the present study. We obtained blood samples from healthy adult donors with the approval of the ethics committee. Peripheral blood mononuclear cells (PBMCs) were stimulated with CM-Exs in the absence or presence of interleukin-2 (IL-2) and IL-12. Cell surface markers and intracellular cytokine production were analysed by flow cytometry. CM-Ex stimulation enhanced the expression of CD69 on NK cells. Although CM-Ex stimulation alone did not induce interferon-γ (IFN-γ) production by NK cells or γδT cells, simultaneous stimulation with CM-Ex, IL-2 and IL-12 significantly enhanced IFN-γ production. In conclusion, cow milk consumption alone may not activate immune cells; however, CM-Exs could enhance immune cells under inflammatory conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/25785826.2020.1791400DOI Listing
December 2020

Possible involvement of DNA methylation in hippocampal synaptophysin gene expression during postnatal development of mice.

Neurochem Int 2020 01 4;132:104587. Epub 2019 Nov 4.

Laboratory of Animal Genetics and Physiology, Department of Animal Science, College of Bioresource Sciences, Nihon University, Japan.

Synaptophysin (Syp) is an integral membrane protein of synaptic vesicles, and is ubiquitously expressed in neurons throughout the brain. As Syp expression is correlated with synaptogenesis during development of the central nervous system, the expression of Syp is considered to be a critical aspect of neuronal maturation and circuit formation. However, little information is available concerning the regulatory mechanisms of Syp gene expression during postnatal development of the brain. In the present study, we investigated changes in Syp mRNA in the hippocampus of mice during postnatal development, and examined the gene regulation mechanisms, focusing on DNA methylation. We found that hippocampal Syp expression involving both mRNA and protein levels increased during the first two weeks of life, and that this increase was accompanied by a transition from hypermethylation to hypomethylation at the CpG sites of the Syp gene upstream region. In addition, DNA demethylating agent 5-Aza-2'-deoxycytidine (5-aza-dC) de-repressed Syp gene expression both in vitro in Neuro-2a mouse neuronal cells and in vivo in the hippocampus of early postnatal mice. Furthermore, the methylation levels at upstream region of Syp gene in the hippocampus of developing mice was decreased by intraperitoneal injection of 5-aza-dC. These results suggest that Syp gene regulation, at least during postnatal brain development, could be mediated by DNA methylation. Our findings promote understanding of the molecular basis of synaptogenesis during postnatal brain development, and provide novel insight into therapeutic aspects of neurodevelopmental disorders involving synaptic dysfunction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuint.2019.104587DOI Listing
January 2020

Lactoferrin promotes autophagy via AMP-activated protein kinase activation through low-density lipoprotein receptor-related protein 1.

Biochem Biophys Res Commun 2017 11 1;493(1):509-513. Epub 2017 Sep 1.

Laboratory of Animal Genetics and Physiology, Department of Animal Science, College of Bioresource Sciences, Nihon University, Japan. Electronic address:

Lactoferrin (LF) is a multifunctional, iron-binding glycoprotein in mammalian secretions, such as breast milk, and has several beneficial effects for human health. However, how these effects are exerted at the cellular level is still largely unknown. In this study, we investigated the effects of LF on autophagy activity in NIH/3T3 mouse fibroblasts. LF from bovine milk was found to increase LC3-I to LC3-II conversion and LC3-positive cytosolic punctate structures because of increased autophagy flux. Knockdown of the putative LF receptor low-density receptor-related protein 1 (LRP1) completely abolished LC3 conversion in cells by LF treatment. Moreover, exposure to LF increased the phosphorylation levels of AMPK in cells, and treatment of dorsomorphin, a pharmacological inhibitor of AMPK signaling, attenuated LC3 conversion by LF. Therefore, we concluded that the beneficial effects of LF might be due to an increase of autophagy activity via AMPK signaling through the LRP1 receptor. These findings provide a novel insight into the physiological role of LF for the maintenance of cellular and tissue homeostasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2017.08.160DOI Listing
November 2017

Transgenic Monkey Model of the Polyglutamine Diseases Recapitulating Progressive Neurological Symptoms.

eNeuro 2017 Mar-Apr;4(2). Epub 2017 Mar 28.

Department of Neurophysiology, National Institute of Neuroscience National Center of Neurology and Psychiatry , Tokyo 187-8502, Japan.

Age-associated neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and the polyglutamine (polyQ) diseases, are becoming prevalent as a consequence of elongation of the human lifespan. Although various rodent models have been developed to study and overcome these diseases, they have limitations in their translational research utility owing to differences from humans in brain structure and function and in drug metabolism. Here, we generated a transgenic marmoset model of the polyQ diseases, showing progressive neurological symptoms including motor impairment. Seven transgenic marmosets were produced by lentiviral introduction of the human ataxin 3 gene with 120 CAG repeats encoding an expanded polyQ stretch. Although all offspring showed no neurological symptoms at birth, three marmosets with higher transgene expression developed neurological symptoms of varying degrees at 3-4 months after birth, followed by gradual decreases in body weight gain, spontaneous activity, and grip strength, indicating time-dependent disease progression. Pathological examinations revealed neurodegeneration and intranuclear polyQ protein inclusions accompanied by gliosis, which recapitulate the neuropathological features of polyQ disease patients. Consistent with neuronal loss in the cerebellum, brain MRI analyses in one living symptomatic marmoset detected enlargement of the fourth ventricle, which suggests cerebellar atrophy. Notably, successful germline transgene transmission was confirmed in the second-generation offspring derived from the symptomatic transgenic marmoset gamete. Because the accumulation of abnormal proteins is a shared pathomechanism among various neurodegenerative diseases, we suggest that this new marmoset model will contribute toward elucidating the pathomechanisms of and developing clinically applicable therapies for neurodegenerative diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/ENEURO.0250-16.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5368386PMC
October 2017

Lysosomal membrane protein SIDT2 mediates the direct uptake of DNA by lysosomes.

Autophagy 2017 Jan 15;13(1):218-222. Epub 2016 Nov 15.

a Department of Degenerative Neurological Diseases , National Institute of Neuroscience, National Center of Neurology and Psychiatry , Kodaira, Tokyo , Japan.

Lysosomes degrade macromolecules such as proteins and nucleic acids. We previously identified 2 novel types of autophagy, RNautophagy and DNautophagy, where lysosomes directly take up RNA and DNA, in an ATP-dependent manner, for degradation. We have also reported that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference defective-1), mediates RNA translocation during RNautophagy. In this addendum, we report that SIDT2 also mediates DNA translocation in the process of DNautophagy. These findings help elucidate the mechanisms underlying the direct uptake of nucleic acids by lysosomes and the physiological functions of DNautophagy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15548627.2016.1248019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5245770PMC
January 2017

Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes.

Autophagy 2016 ;12(3):565-78

a Department of Degenerative Neurological Diseases , National Institute of Neuroscience, National Center of Neurology and Psychiatry , Kodaira , Tokyo , Japan.

Lysosomes are thought to be the major intracellular compartment for the degradation of macromolecules. We recently identified a novel type of autophagy, RNautophagy, where RNA is directly taken up by lysosomes in an ATP-dependent manner and degraded. However, the mechanism of RNA translocation across the lysosomal membrane and the physiological role of RNautophagy remain unclear. In the present study, we performed gain- and loss-of-function studies with isolated lysosomes, and found that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference deficient-1), mediates RNA translocation during RNautophagy. We also observed that SIDT2 is a transmembrane protein, which predominantly localizes to lysosomes. Strikingly, knockdown of Sidt2 inhibited up to ˜50% of total RNA degradation at the cellular level, independently of macroautophagy. Moreover, we showed that this impairment is mainly due to inhibition of lysosomal RNA degradation, strongly suggesting that RNautophagy plays a significant role in constitutive cellular RNA degradation. Our results provide a novel insight into the mechanisms of RNA metabolism, intracellular RNA transport, and atypical types of autophagy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15548627.2016.1145325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836006PMC
December 2016

Valproate administration to mice increases hippocampal p21 expression by altering genomic DNA methylation.

Neuroreport 2015 Oct;26(15):915-20

Laboratory of Animal Genetics and Physiology, Department of Animal Science, College of Bioresource Sciences, Nihon University, Kanagawa, Japan.

Although valproate (VPA) is used widely in the treatment of bipolar mood disorder and epilepsy, the precise mechanism of action in the brain remains elusive. In this study, we investigated the effects of subchronic VPA administrations on the expression of the cyclin-dependent kinase inhibitor (Cdkn) family in the hippocampus of adult mice. The administration of VPA specifically increased hippocampal p21 expression involving both mRNA and protein levels, but other members of the Cdkn family were not affected. We identified two CpG islands in the p21 gene regulatory region, located distal and proximal to the transcription start site. VPA altered genomic DNA methylation patterns in the distal region, but not in the proximal promoter region. However, no change was found in DNA methyltransferase (Dnmt) 1 or Dnmt3a protein levels, suggesting an involvement in active demethylation mechanisms. These findings suggest that VPA alters the gene expression of cell cycle regulators by modulating promoter DNA methylation, and this resulted in altered hippocampal cell proliferation. These findings promote understanding of the actions of VPA in the brain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/WNR.0000000000000448DOI Listing
October 2015

RNautophagy/DNautophagy possesses selectivity for RNA/DNA substrates.

Nucleic Acids Res 2015 Jul 1;43(13):6439-49. Epub 2015 Jun 1.

Department of Degenerative Neurological Disease, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan

Lysosomes can degrade various biological macromolecules, including nucleic acids, proteins and lipids. Recently, we identified novel nucleic acid-degradation systems termed RNautophagy/DNautophagy (abbreviated as RDA), in which RNA and DNA are directly taken up by lysosomes in an ATP-dependent manner and degraded. We also found that a lysosomal membrane protein, LAMP2C, the cytoplasmic region of which binds to RNA and DNA, functions, at least in part, as an RNA/DNA receptor in the process of RDA. However, it has been unclear whether RDA possesses selectivity for RNA/DNA substrates and the RNA/DNA sequences that are recognized by LAMP2C have not been determined. In the present study, we found that the cytosolic region of LAMP2C binds to poly-G/dG, but not to poly-A/dA, poly-C/dC, poly-dT or poly-U. Consistent with this binding activity, poly-G/dG was transported into isolated lysosomes via RDA, while poly-A/dA, poly-C/dC, poly-dT and poly-U were not. GGGGGG or d(GGGG) sequences are essential for the interaction between poly-G/dG and LAMP2C. In addition to poly-G/dG, G/dG-rich sequences, such as a repeated GGGGCC sequence, interacted with the cytosolic region of LAMP2C. Our findings indicate that RDA does possess selectivity for RNA/DNA substrates and that at least some consecutive G/dG sequence(s) can mediate RDA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkv579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4513860PMC
July 2015

Age-dependent sensitivity to glucocorticoids in the developing mouse basolateral nucleus of the amygdala.

Psychoneuroendocrinology 2014 Aug 19;46:64-77. Epub 2014 Apr 19.

Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan; CREST, Japan Science and Technology Agency, 4-1-8 Hon-machi, Kawaguchi, Saitama 322-0012, Japan. Electronic address:

Experiences of severe trauma during childhood are thought to be risk factors for developing mental disorders, such as anxiety and mood disorders, later in life. Correspondingly, exposure of rodents to early-life stress has been shown to affect neuronal circuitry and emotional behavior in adulthood, indicating a significant impact of stress on brain development. One current hypothesis proposes that the developing central nervous system is more sensitive to environmental influences, such as stress, than the adult. To test this hypothesis, we compared long-lasting effects of systemic corticosterone (CORT) administrations in two distinct early developmental periods. Mice exposed to early-neonatal CORT treatment on postnatal days (PD) 2-4 exhibited strongly enhanced excitability of neurons of the basolateral nucleus of the amygdala (BLA) in early adolescence and displayed impaired extinction of contextually conditioned fear memory, a type of behavior in which the BLA plays an important role. Furthermore, gene-expression of NMDA receptor subunits as well as calcium-activated K(+)-channels was reduced in the amygdala. In contrast, exposure to the same CORT concentrations in a late-neonatal period (PD17-19) did not significantly affect BLA electrophysiology or extinction learning in adolescence. These results suggest age-dependent consequences of neonatal CORT exposure in amygdala neurons and provide evidence for a detrimental influence of early-neonatal stress on adolescent fear-memory processing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.psyneuen.2014.04.007DOI Listing
August 2014

Direct uptake and degradation of DNA by lysosomes.

Autophagy 2013 Aug 21;9(8):1167-71. Epub 2013 May 21.

Department of Degenerative Neurological Diseases; National Institute of Neuroscience; National Center of Neurology and Psychiatry; Kodaira, Tokyo Japan; Department of Electrical Engineering and Bioscience; Graduate School of Advanced Science and Engineering; Waseda University; Shinjuku, Tokyo Japan.

Lysosomes contain various hydrolases that can degrade proteins, lipids, nucleic acids and carbohydrates. We recently discovered "RNautophagy," an autophagic pathway in which RNA is directly taken up by lysosomes and degraded. A lysosomal membrane protein, LAMP2C, a splice variant of LAMP2, binds to RNA and acts as a receptor for this pathway. In the present study, we show that DNA is also directly taken up by lysosomes and degraded. Like RNautophagy, this autophagic pathway, which we term "DNautophagy," is dependent on ATP. The cytosolic sequence of LAMP2C also directly interacts with DNA, and LAMP2C functions as a receptor for DNautophagy, in addition to RNautophagy. Similarly to RNA, DNA binds to the cytosolic sequences of fly and nematode LAMP orthologs. Together with the findings of our previous study, our present findings suggest that RNautophagy and DNautophagy are evolutionarily conserved systems in Metazoa.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/auto.24880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3748189PMC
August 2013

Discovery of a novel type of autophagy targeting RNA.

Autophagy 2013 Mar 4;9(3):403-9. Epub 2013 Jan 4.

Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan.

Regulated degradation of cellular components by lysosomes is essential to maintain biological homeostasis. In mammals, three forms of autophagy, macroautophagy, microautophagy and chaperone-mediated autophagy (CMA), have been identified. Here, we showed a novel type of autophagy, in which RNA is taken up directly into lysosomes for degradation. This pathway, which we term "RNautophagy," is ATP-dependent, and unlike CMA, is independent of HSPA8/Hsc70. LAMP2C, a lysosomal membrane protein, serves as a receptor for this pathway. The cytosolic tail of LAMP2C specifically binds to almost all total RNA derived from mouse brain. The cytosolic sequence of LAMP2C and its affinity for RNA are evolutionarily conserved from nematodes to humans. Our findings shed light on the mechanisms underlying RNA homeostasis in higher eukaryotes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/auto.23002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3590259PMC
March 2013

Early milk availability modulates the activity of choline acetyltransferase in the cerebral cortex of rats.

Anim Sci J 2011 Oct 16;82(5):684-8. Epub 2011 Jun 16.

Department of Animal Science, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa, Japan.

The purpose of the present study was to investigate the effect of milk in the early stage of lactation on the maturation of cholinergic neurons in the cerebral cortex of rats. Pups were removed from their mothers immediately following parturition and placed with foster dams at days 5-7 of lactation. At days 18 and 56 after birth, the activity of choline acetyltransferase (ChAT), an enzyme responsible for acetylcholine synthesis, in different areas of the cerebral cortex was examined by high-performance liquid chromatography electrochemical detection. In the frontal and hindlimb/parietal regions of the cerebral cortex, the lack of early milk significantly decreased ChAT activity at days 18 and 56. There was no effect on gains in the body or brain weight of infants. ChAT activity in the occipital area tended to be lower in the early milk-deprived rats. The intake of early milk potentially contributes not only to nutrients for the growth of newborn infants, but also to the functional maturation of the cholinergic neurotransmission system in a region-specific manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1740-0929.2011.00892.xDOI Listing
October 2011

Asymmetric regulation by estrogen at the cholinergic gene locus in differentiated NG108-15 neuronal cells.

Life Sci 2010 Jun 27;86(23-24):839-43. Epub 2010 Mar 27.

Department of Animal Science, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252-8510, Japan.

Aims: Estrogen acts as a neurogenerative and neuroprotective factor in the cholinergic system. Choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are regarded as markers of cholinergic neurons. The genes coding these proteins are located at a common locus, the cholinergic gene locus. However, few details concerning activation of the locus have been obtained. We examined the effect of estrogen on the activation pattern of the locus using a cholinergic cell line.

Main Methods: NG108-15 neuronal cells, as a model of cholinergic neurons, were used. Dose-dependent effects of estradiol (E2) on the gene expression of ChAT and VAChT were quantitatively determined by a real-time RT-PCR. The expression of ChAT mRNA variants was qualitatively evaluated by RT-PCR using specific primers.

Key Findings: The expression of ChAT and VAChT mRNA was strongly enhanced with the induction of differentiation. The enhanced expression of ChAT mRNA was further increased dose-dependently by E2 (10(-10) to 10(-7)M), while that of VAChT mRNA did not respond to E2. The up-regulation of ChAT mRNA expression by E2 was abolished by co-treatment with a pure-antagonist of estrogen receptors. A qualitative analysis of ChAT mRNA variants revealed the R types, which share a common sequence with the VAChT gene, and type M ChAT mRNA to mainly be expressed, and that the appearance of these variants was not altered by E2.

Significance: The cholinergic gene locus in differentiated NG108-15 neuronal cells is further activated by E2, but the effect is restricted to the transcription of ChAT gene.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.lfs.2010.03.014DOI Listing
June 2010

Involvement of histone acetylation in the regulation of choline acetyltransferase gene in NG108-15 neuronal cells.

Neurochem Int 2010 Mar 25;56(4):627-33. Epub 2010 Jan 25.

Department of Animal Science, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252-8510, Japan.

Post-translational modification of histone such as acetylation of N-terminal of lysine residues influences gene expression by modulating the accessibility of specific transcription factors to the promoter region, and is essential for a wide variety of cellular processes in the development of individual tissues, including the brain. However, few details concerning the acquisition of specific neurotransmitter phenotype have been obtained. In the present study, we investigated the possible involvement of histone acetylation in the gene expression of choline acetyltransferase (ChAT), a specific marker for cholinergic neuron and its function, in NG108-15 neuronal cells as an in vitro model of cholinergic neuron. Treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA), which induces global histone hyper-acetylation of the cells, resulted in marked increase in the expression of ChAT gene in proliferating NG108-15 cells. Furthermore, RT-PCR analysis using primer pairs for individual variants of ChAT mRNA (R1-4, N1, and M type) revealed that M type, not R1-4 and N1 type, ChAT mRNA were mainly transcribed, and chromatin immunoprecipitation assay indicated that the promoter region of M type ChAT gene was highly acetylated, in the dibutyryl cyclic AMP-induced neuronal differentiation of NG108-15 cells. The present findings demonstrate that the acquisition of neurotransmitter phenotype is epigenetically, at least the hyper-acetylation on the core promoter region of ChAT gene, regulated in NG108-15 neuronal cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuint.2010.01.007DOI Listing
March 2010

Induction of cholinergic differentiation by 5-azacytidine in NG108-15 neuronal cells.

Neuroreport 2009 Jan;20(2):157-60

Department of Animal Science, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa, Japan.

The DNA-demethylating agent 5-azacytidine (5-azaC) causes extensive genomic demethylation of 5-methyl-cytosine residues and reduces DNA methyltransferase activity in cells. This study evaluated the effect of 5-azaC on neuronal differentiation in proliferating NG108-15 neuronal cells, which exhibit cholinergic traits. The expression of choline acetyltransferase, an enzyme responsible for acetylcholine synthesis, was increased at both the mRNA and protein level, and neurite outgrowth was markedly induced with an increase of neurofilament-heavy chain protein, in the 5-azaC-treated cells. These findings show that global DNA demethylation markedly induces the expression of the neurotransmitter phenotype and morphological differentiation in NG108-15 neuronal cells as a model for cholinergic neuron.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/WNR.0b013e32831d28eeDOI Listing
January 2009

Estradiol regulates alternative splicing of estrogen receptor-alpha mRNA in differentiated NG108-15 neuronal cells.

Life Sci 2008 Mar 12;82(13-14):692-8. Epub 2008 Jan 12.

Department of Animal Science, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan.

The biological actions of estrogen are mostly conveyed through interaction with two different types of estrogen receptor (ER), ER-alpha and ER-beta. With regard to ER-alpha, an alternatively spliced form and its translated product, truncated estrogen receptor product-1 (TERP-1), have been identified in the rat pituitary. TERP-1 has the ability to inhibit the ER binding to DNA response element by forming hetero-dimers with the wild-type ER. Furthermore, TERP-1 expression increased concurrently with serum estrogen levels. Although estrogen also plays important roles in the central nervous system, the existence and regulatory mechanism of alternatively spliced ER-alpha mRNA expression has remained unclear. The present study evaluated the expression of the alternatively spliced form of the ER-alpha gene, and examined the influence of a representative ER ligand, 17beta-estradiol (E2), on the expression in differentiated NG108-15 neuronal cells. A real-time RT-PCR analysis using primer sets designed to amplify from exons 3 to 4, exons 4 to 5, exons 5 to 6, exons 6 to 7, and exons 7 to 8 of the mouse ER-alpha gene revealed the existence of alternatively spliced ER-alpha mRNA and its putative transcription initiation site, located between exon 4 and exon 5. Although E2 had no apparent effect on the overall expression of ER-alpha mRNA, it reduced the incidence of the alternatively spliced form of ER-alpha. The down-regulation by E2 predominantly arose via binding to nuclear ERs. The present study demonstrated that alternatively spliced ER-alpha mRNA is expressed in differentiated NG108-15 neuronal cells, and provides evidence for the functional up-regulation of ER-alpha via the ligand-binding activation of ERs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.lfs.2008.01.001DOI Listing
March 2008

Characterization of cell death induced by ethacrynic acid in a human colon cancer cell line DLD-1 and suppression by N-acetyl-L-cysteine.

Cancer Sci 2003 Oct;94(10):886-93

Second Department of Biochemistry, Hirosaki University School of Medicine, Hirosaki, Aomori 036-8562, Japan.

Since ethacrynic acid (EA), an SH modifier as well as glutathione S-transferase (GST) inhibitor, has been suggested to induce apoptosis in some cell lines, its effects on a human colon cancer cell line DLD-1 were examined. EA enhanced cell proliferation at 20-40 microM, while it caused cell death at 60-100 microM. Caspase inhibitors did not block cell death and DNA ladder formation was not detected. Poly(ADP-ribose) polymerase, however, was cleaved into an 82-kDa fragment, different from an 85-kDa fragment that is specific for apoptosisis. The 82-kDa fragment was not recognized by antibody against PARP fragment cleaved by caspase 3. N-Acetyl-L-cysteine (NAC) completely inhibited EA-induced cell death, but 3(2)-t-butyl-4-hydroxyanisole or pyrrolidinedithiocarbamate ammonium salt did not. Glutathione (GSH) levels were dose-dependently increased in cells treated with EA and this increase was hardly affected by NAC addition. Mitogen-activated protein kinase (MAPK) kinase (MEK) 1, extracellular signal-regulated kinase (ERK) 1 and GST P1-1 were increased in cells treated with 25-75 microM EA, while c-Jun N-terminal kinase (JNK) 1 and p38 MAPK were markedly decreased by 100 microM EA. NAC repressed EA-induced alterations in these MAPKs and GST P1-1. p38 MAPK inhibitors, SB203580 and FR167653, dose-dependently enhanced EA-induced cell death. An MEK inhibitor, U0126, did not affect EA-induced cell death. These studies revealed that EA induced cell death concomitantly with a novel PARP fragmentation, but without DNA fragmentation. p38 MAPK was suggested to play an inhibitory role in EA-induced cell death.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1349-7006.2003.tb01371.xDOI Listing
October 2003

Polymorphic glutathione S-transferase subunit 3 of rat liver exhibits different susceptibilities to carbon tetrachloride: differences in their interactions with heat-shock protein 90.

Biochem J 2003 Jun;372(Pt 2):611-6

Second Department of Biochemistry, Hirosaki University School of Medicine, 5 Zaifu-cho, Japan.

Rat glutathione S-transferase (GST) subunit 3 gene has polymorphism, one type encoding Asn(198)-Cys(199) (NC type) and another encoding Lys(198)-Ser(199) (KS type). To examine whether the two types of GST 3-3 exhibit different susceptibilities to oxidative stress in vivo, rats were administered with CCl(4), a hepatotoxin causing severe oxidative stress, and its effect on liver GST 3-3 was compared. Decrease in GST activities in liver due to CCl(4) administration was more evident in NC type rats than in KS type rats, and most GST activities of KS type rats were confined to S-hexylglutathione-Sepharose, whereas those of NC type rats were not. Decreases in GST subunits 1 and 3 were more marked in NC type rats and glutathiolated NC type GST 3-3 was also detected. These results indicated that KS and NC type GST 3-3 of rat livers exhibited different susceptibilities to CCl(4) in vivo. A protein consisting of a subunit with molecular mass of 90 kDa was shown to bind to KS type GST 3-3 but not to NC type. This protein was identified as heat-shock protein (HSP) 90beta by N-terminal amino acid sequencing and immunoblotting. A specific HSP90 inhibitor geldanamycin released their binding. There was no difference in the binding of apoptosis signal-regulating kinase 1 to GST 3-3 between NC and KS type rats. These findings suggest that HSP90 interacts with KS type GST 3-3 and thereby protects it from inactivation due to CCl(4).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20021788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223406PMC
June 2003

Transcriptional activation of the MUC2 gene by p53.

J Biol Chem 2002 Dec 8;277(50):48270-5. Epub 2002 Oct 8.

Second Department of Biochemistry, Hirosaki University School of Medicine, Zaifucho 5, Aomori 036-8562, Japan.

MUC2 is one of the major components of mucins that provide a protective barrier between epithelial surfaces and the gut lumen. We investigated possible alterations of MUC2 gene expression by p53 and p21(Sdi1/Waf1/Cip1) in a human colon cancer cell line, DLD-1, establishing subclones in which a tetracycline-regulatable promoter controls exogenous p53 and p21 expression. MUC2 mRNA more significantly increased in response to p53 than to p21. Unexpectedly, MUC2 expression was also induced in human osteosarcoma cells, U-2OS and Saos-2, by exogenous p53. We next performed a reporter assay to test the direct regulation of MUC2 gene expression by p53. Deletion and mutagenesis of the MUC2 promoter region showed that it contains two sites for transactivation by p53. Furthermore, an electrophoretic mobility shift assay indicated that p53 binds to those elements. We analyzed MUC2 expression in other cell types possessing a functional p53 after exposure to various forms of stress. In MCF7 breast cancer and A427 lung cancer cells, MUC2 expression was increased along with the endogenous p53 level by actinomycin D, UVC, and x-ray, but not in RERF-LC-MS lung cancer cells carrying a mutated p53. These results suggest that p53 directly activates the MUC2 gene in many cell types.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M207986200DOI Listing
December 2002