Yasuyuki Irie

Arcadlin Is a Neural Activity-regulated Cadherin Involved in Long Term Potentiation

Neural activity results in long term changes that underlie synaptic plasticity. To examine the molecular basis of activity-dependent plasticity, we have used differential cloning techniques to identify genes that are rapidly induced in brain neurons by synaptic activity. Here, we identify a novel cadherin molecule Arcadlin (activity-regulated cadherin-like protein).arcadlin mRNA is rapidly and transiently induced in hippocampal granule cells by seizures and byN-methyl-d-aspartate-dependent synaptic activity in long term potentiation. The extracellular domain of Arcadlin is most homologous to protocadherin-8; however, the cytoplasmic region is distinct from that of any cadherin family member. Arcadlin protein is expressed at the synapses and shows a homophilic binding activity in a Ca2+-dependent manner. Furthermore, application of Arcadlin antibody reduces excitatory postsynaptic potential amplitude and blocks long term potentiation in hippocampal slices. Its close homology with cadherins, its rapid inducibility by neural activity, and its involvement in synaptic transmission suggest that Arcadlin may play an important role in activity-induced synaptic reorganization underlying long term memory.

Molecular Cloning and Characterization of Amida, a Novel Protein Which Interacts with a Neuron-specific Immediate Early Gene Product Arc, Contains Novel Nuclear Localization Signals, and Causes Cell Death in Cultured Cells

Amida was isolated by the yeast two-hybrid system as a novel protein which associated with Arc, a non-transcriptional immediate early gene specific to the brain. Amida was confirmed to be associated with Arc in vitro and in vivo. Amida shows no homology to known proteins. Amida is ubiquitously expressed, although it is abundant in the brain. A transfection study revealed that Amida was localized in the nucleus and after 72 h the transfected cells underwent apoptosis. Furthermore, we found two nuclear localization signals and a domain needed for interacting with Arc was encompassed by two nuclear localization signals. Co-transfection experiment with Amida and Arc suggested that Amida transported Arc into the nucleus and negatively regulated Amida-induced cell death. These results indicate that Arc together with Amida may modulate cell death in the brain.

Calcineurin potentiates the activation of procaspase-3 by accelerating its proteolytic maturation.

We have previously shown that procaspase-3 exists in a high molecular weight complex in neonatal rat brain. Here, we purify and identify the protein that interacts with procaspase-3 from rat neonatal cortex. We searched binding proteins to procaspase-3 from a cytosolic extract of neonatal rat brain using chromatogram, two-dimensional gel electrophoresis, and far Western immunoblot. Analysis by tandem mass spectrometry identified the protein as a regulatory subunit of calcineurin (calcineurin B). Overexpression of calcineurin B in HEK293 cells potentiated processing of caspase-3 and apoptosis triggered by tumor necrosis factor-α and cycloheximide treatment. In a cell-free system, overexpression of calcineurin B in HEK293 cells markedly increased processing of caspase-3 by cytochrome c. Immunodepletion of calcineurin B from cytosolic extracts from Jurkat cells decreased processing of caspase-3 by cytochrome c. Knockdown of calcineurin B by RNA interference resulted in reduced apoptosis in HEK293 cells but not in caspase-3-deficient MCF-7 cells. These results suggest that calcineurin B potentiates the activation of procaspase-3 by accelerating its proteolytic maturation.

Molecular cloning of novel Monad binding protein containing tetratricopeptide repeat domains

MINT‐6551090: Monad (uniprotkb:Q96MX6) physically interacts (MI:0218) with RPAP3 (uniprotkb:Q9H6T3) by anti tag coimmunoprecipitation (MI:0007) MINT‐6551101, MINT‐6551118: Monad (uniprotkb:Q96MX6) physically interacts (MI:0218) with RPAP3 (uniprotkb:Q9H6T3) by pull down (MI:0096) MINT‐6551132: RPAP3 (uniprotkb:Q9H6T3) physically interacts (MI:0218) with Monad (uniprotkb:Q96MX6) by anti bait coimmunoprecipitation (MI:0006)

Involvement of a single-stranded DNA binding protein, ssCRE-BP/Purα, in morphine dependence

We have purified a nuclear protein from mouse cerebella that binds to single‐stranded oligo‐DNA of cAMP response element and is modulated by morphine treatment. Isolation of the cDNA clone showed that the nuclear protein (ssCRE‐BP) was identical to Purα, a DNA binding protein for single‐stranded purine‐rich sequences that was originally isolated as a replication factor. ssCRE‐BP/Purα and mRNA were abundant in the brain. The levels of ssCRE‐BP/Purα and the transcript were not changed by chronic morphine treatment, however, the levels of an activator of ssCRE‐BP/Purα, which is necessary for the DNA binding, may be modulated by the treatment.

CHARACTERIZATION OF A NUCLEAR FACTOR THAT ENHANCES DNA BINDING ACTIVITY OF ssCRE-BP/PURα, A SINGLE-STRANDED DNA BINDING PROTEIN

Pur alpha has been identified as a single-stranded DNA binding protein that specifically binds to the purine-rich strand present in the DNA replication initiation zone of the human c-myc gene. We have previously demonstrated that chronic morphine treatment decreases the DNA binding activity of ssCRE-BP (single-stranded cyclic AMP response element-binding protein), which has been shown to be identical to pur alpha by cDNA cloning, and is abundant in the brain. In this report we identified an activator of ssCRE-BP/pur alpha in the brain and characterized it. Although purified ssCRE-BP/pur alpha or its GST-fusion protein exhibited very low DNA binding activities, they were markedly enhanced by including nuclear extract in the binding assay. The enhanced binding activity is trypsin-sensitive, heat-stable and has a molecular weight of approximately 66 kDa. Casein could substitute for the activator and increased the DNA binding activity of ssCRE-BP/pur alpha by one order. A series of deletion mutants were prepared in order to determine the DNA binding and activator interacting domains, and both of them were found to reside in AA 50-215 of ssCRE-BP/pur alpha. These data suggest that the DNA binding activity of ssCRE-BP/pur alpha is augmented by a nuclear protein, which may modulate the ssCRE-BP/pur alpha activity to develop morphine dependence and tolerance.

RPAP3 splicing variant isoform 1 interacts with PIH1D1 to compose R2TP complex for cell survival.

We previously characterized RNA polymerase II-associated protein 3 (RPAP3) as a cell death enhancer. Here we report the identification and characterization of splicing isoform of RPAP3, isoform 1 and 2. We investigated the interaction between RPAP3 and PIH1 domain containing protein 1 (PIH1D1), and found that RPAP3 isoform 1, but not isoform 2, interacted with PIH1D1. Furthermore, knockdown of RPAP3 isoform 1 by small interfering RNA down-regulated PIH1D1 protein level without affecting PIH1D1 mRNA. RPAP3 isoform 2 potentiated doxorubicin-induced cell death in human breast cancer T-47 cells although isoform 1 showed no effect. These results suggest that R2TP complex is composed of RPAP3 isoform 1 for its stabilization, and that RPAP3 isoform 2 may have a dominant negative effect on the survival potency of R2TP complex.

Inflammatory cytokines decrease the expression of nicotinic acetylcholine receptor during the cell maturation

It is known that the nervous system significantly attenuates systemic inflammatory responses through the parasympathetic nervous system. Furthermore, it has been reported that the alpha7 subunit of a nicotinic acetylcholine receptor is required for a cholinergic inhibition against cytokine synthesis in a macrophage. As antigen-presenting cells (APCs) play a central role in the generation of primary T cell responses and the maintenance of immunity, in this study, we investigated the expression level of nicotinic receptors of a p53-deficient APC cell line (JawsII) derived from a mouse bone marrow. We showed that stimulation of the JawsII cells with lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-α) led increase of CD80 and CD86 expression while diminishment of the surface nicotinic receptor. On the other hand, stimulation of nicotinic receptor had no effect on these phenomena. Furthermore, we examined the ability of the cells to release cytokine when stimulated with both nicotine and LPS and showed that the stimulation with LPS augmented the secretion of IL-1a, IL-1b, IL-6, and TNF-α. These results suggested that nicotinic stimulation had no effect on the diminishment of alpha7 nicotinic acetylcholine receptor on JawsII cells by LPS stimulation.

Methamphetamine induces endoplasmic reticulum stress related gene CHOP/Gadd153/ddit3 in dopaminergic cells

We examined the toxicity of methamphetamine and dopamine in CATH.a cells, which were derived from mouse dopamine-producing neural cells in the central nervous system. Use of the quantitative real-time polymerase chain reaction revealed that transcripts of the endoplasmic reticulum stress related gene (CHOP/Gadd153/ddit3) were considerably induced at 24–48 h after methamphetamine administration (but only under apoptotic conditions), whereas dopamine slightly induced CHOP/Gadd153/ddit3 transcripts at an early stage. We also found that dopamine and methamphetamine weakly induced transcripts for the glucose-regulated protein 78 gene (Grp78/Bip) at the early stage. Analysis by immunofluorescence microscopy demonstrated an increase of　CHOP/Gadd153/ddit3 and Grp78/Bip proteins at 24 h after methamphetamine administration. Treatment of CATH.a cells with methamphetamine caused a re-distribution of dopamine inside the cells, which mimicked the presynaptic activity of neurons with cell bodies located in the ventral tegmental area or the substantia nigra. Thus, we have demonstrated the existence of endoplasmic reticulum stress in a model of presynaptic dopaminergic neurons for the first time. Together with the recent evidence suggesting the importance of presynaptic toxicity, our findings provide new insights into the mechanisms of dopamine toxicity, which might represent one of the most important mechanisms of methamphetamine toxicity and addiction.

Analysis of the Tβγ-binding domain of MEKA/phosducin

MEKA/phosducin, a 33 kDa phosphoprotein in the photoreceptor cell, associates with transducin beta gamma (T beta gamma) with its N-terminal domain (N-terminal 105 amino acids of MEKA), and translocates T beta gamma from the photoreceptor disc membrane to the soluble fraction. The present study further localized the T beta gamma-binding domain to aa 17-105 of MEKA, and showed that the activity of MEKA to translocate T beta gamma depends on the domain. A series of deletion mutant MEKA proteins were prepared to investigate the domain of MEKA which binds to and translocates T beta gamma. Both binding and translocation activities were not impaired by the deletion of the N-terminal 16 amino acids of MEKA, but completely abolished by further deletion to 42Val. Although anti-MEKA serum inhibited the T beta gamma-MEKA association, the antiserum absorbed with a recombinant peptide corresponding to aa 17-105 of MEKA did not, confirming that aa 17-105 of MEKA directly interacts with T beta gamma.