Hiroyuki Kato

Functional coupling of the metabotropic glutamate receptor, InsP3 receptor and L-type Ca2+ channel in mouse CA1 pyramidal cells

•  While the metabotropic glutamate receptor (mGluR) is supposed to modulate L‐type voltage‐dependent calcium channels (L‐VDCCs), its reported actions include both facilitation and suppression, and thus the modulation of L‐VDCCs by synaptic activity has still been under debate. •  In this study, using acute hippocampal slices of subtype‐specific knockout mice, we have shown that mGluR5 induces facilitation of the depolarization‐evoked calcium current. •  This facilitation was not accompanied by the change in single‐channel properties of the L‐VDCC itself, but required the activation of calcium‐induced calcium release that was triggered by L‐VDCC opening. •  L‐VDCCs and mGluR5 were shown to form a complex by coimmunoprecipitation, suggesting that the specific functional coupling between mGluR5, InsP3 receptors and L‐VDCCs played a pivotal role in the calcium‐current facilitation. •  Our study has identified a novel mechanism of the interaction between the mGluR and calcium signalling, and suggested a contribution of mGluR5 to synaptic plasticity.

Non-Hebbian synaptic plasticity induced by repetitive postsynaptic action potentials.

Modern theories on memory storage have mainly focused on Hebbian long-term potentiation (LTP), which requires coincident activation of presynaptic and postsynaptic neurons for its induction. In addition to Hebbian LTP, the roles of non-Hebbian plasticity have also been predicted by some neuronal network models. However, still only a few pieces of evidence have been presented for the presence of such plasticity. In this study, we show in mouse hippocampal slices that LTP can be induced by postsynaptic repetitive depolarization alone in the absence of presynaptic inputs. The induction was dependent on voltage-dependent calcium channels instead of NMDA receptors (NMDARs), whereas the expression mechanism was shared with conventional NMDAR-dependent LTP. During the potentiation, the amplitude of spontaneous EPSCs was increased, suggesting a novel neuron-wide nature of this form of LTP. Furthermore, we also successfully induced LTP with trains of action potentials, which supported the possible existence of depolarizing pulse-induced LTP in vivo. Based on these findings, we suggest a model in which neuron-wide LTP works in concert with synapse-specific Hebbian plasticity to help information processing in memory formation.

Isolation and characterization of temperature-sensitiveplc1 mutants of the yeastSaccharomyces cerevisiae

ThePLC1 gene of the yeastSaccharomyces cerevisiae has been discovered to encode a homolog of mammalian phosphoinositide-specific phospholipase C (PLC). Five temperature-sensitiveplc1 mutants were isolated by in vitro mutagenesis with subsequent plasmid shuffling. All of the amino acid substitutions that caused a temperature-sensitive growth phenotype were located in the X or the Y region, both of which are conserved among PLC isoenzymes. The PLC activity of all products of mutantplc1 genes was dramatically lower than that of the wild-type product, indicating that PLC activity itself is important for cell growth. At the restrictive temperature,plc1 mutant cells ceased growth at random times during the cell cycle, a result that suggests thatPLC1 is required at several or all stages of the cell cycle.

Formation of the transcription initiation complex on mammalian rDNA.

Steps for the formation of transcription initiation complex on the human rRNA gene (rDNA) in vitro were analyzed with partially purified transcription factors and RNA polymerase I. The reaction requires at least two factors besides RNA polymerase I for maximal efficiency. Preincubation and short-pulse analyses of the accurate transcripts revealed the following steps. First, the species-dependent factor, designated TFID, bound to the rDNA template, forming a preinitiation complex (PIC-1) which was resistant to a moderate concentration (0.015 to 0.02%) of Sarkosyl. Other factors, designated TFIA and RNA polymerase I, were then added to convert it to the final preinitiation complex PIC-3. This complex incorporated the first two nucleoside triphosphates of the starting site to complete the initiation complex (IC), which was resistant to a high concentration (0.2%) of Sarkosyl. Binding of TFID was rate limiting in the overall initiation reaction in vitro. Together with the kinetics of incorporation, the results are interpreted to mean that TFID, one bound, remains complexed with rDNA together with TFIA as the PIC-2 for many rounds of transcription by RNA polymerase I. Thus, the formation of PIC-2 may be a prerequisite for the stable opening of rDNA for transcription in vivo.

Two highly homologous mouse odorant receptors encoded by tandemly-linked MOR29A and MOR29B genes respond differently to phenyl ethers.

Since the discovery of odorant receptors (ORs) in rodents, most ORs have remained orphan receptors. Even for deorphanized ORs in vitro, their in vivo properties are largely unknown. Here, we report odor response profiles of two highly homologous mouse ORs, MOR29A and MOR29B, both in vivo and in vitro. The BAC transgenic mouse was generated, in which olfactory sensory neurons (OSNs) expressing the transgenes MOR29A and MOR29B were differently tagged with IRES‐gapECFP and IRES‐gapEYFP, respectively. MOR29A‐ and MOR29B‐expressing OSN axons converged on separate but nearby loci on the dorsal surface of the olfactory bulb (OB). Optical imaging of intrinsic signals in the OB identified five different phenyl ethers as candidate ligands for MOR29B. Based on in vitro calcium imaging with the isolated OSNs and luciferase assay with heterologous cells, only guaiacol and vanillin were found to be potent agonists for MOR29A and MOR29B. Because of its accessible glomerular locations in the dorsal OB and defined odor response profiles both in vivo and in vitro, the MOR29A/29B tagging mouse will serve as an excellent tool for studying both odor‐signal processing and neural circuitry in the OB.

Isocitrate dehydrogenase 1 mutation sensitizes intrahepatic cholangiocarcinoma to the BET inhibitor JQ1

Cholangiocarcinoma is a life‐threatening disease with a poor prognosis. Although genome analysis unraveled some genetic mutation profiles in cholangiocarcinoma, it remains unknown whether such genetic abnormalities relate to the effects of anticancer drugs. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are exclusively found in almost 20% of intrahepatic cholangiocarcinoma (ICC). Recently, the anticancer effects of BET inhibitors including JQ1 have been shown in various tumors. In the present study, we report that the antigrowth effect of JQ1 differs among ICC cells and IDH1 mutation sensitizes ICC cells to JQ1. RBE cells harboring IDH1 mutation was more sensitive to JQ1 than HuCCT1 or HuH28 cells with wild‐type IDH1. JQ1 induced apoptosis only in RBE cells through the upregulation of proapoptotic genes BAX and BIM. We found that the antigrowth effect was not attributed to downregulation of the MYC gene as a well‐known target of JQ1 in various cancer cells. Notably, the forced expression of mutant IDH1 successfully sensitized HuCCT1 cells to JQ1. In addition, AGI‐5198, a selective inhibitor of mutant IDH1 partially reversed the decrease in viability after JQ1 treatment and also suppressed the JQ1‐induced apoptosis in RBE cells. These data suggest that IDH1 mutation contributed to the growth inhibitory effect of JQ1 in RBE cells. Furthermore, given that the effect of mutant IDH1 was not recapitulated in glioblastoma cells, the enhancement of JQ1 sensitivity by IDH1 mutation seems to be specific for ICC cells. Our findings propose a new stratified therapeutic strategy based on IDH1 mutation in ICC.