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JJP Suppl 2006.Fm Lectures Lectures L1 (2PA) L3 (1EA) The ins and outs of glutamate receptor Water and ion channels in kidney function trafficking during synaptic plasticity Sasaki, Sei (Dept. Nephrology. Grad. Sch.Tokyo Med Dent Univ. Nicoll, Roger A. (Department of Cellular and Molecular Tokyo, Japan) Pharmacology, University of California at San Francisco, San Kidney is the main organ in the maintenance of water and electrolyte ho- Francisco, California, USA) meostasis of extracellular fluid of the body. Extensive physiological re- Glutamate, the major excitatory neurotransmitter in the brain, acts pri- search has been performed to understand this important kidney function. marily on two types of ionotropic receptors, AMPA receptors and The research has developed from the organ level to molecular level and NMDA receptors. Work over the past decade indicates that the number the development was accompanied by advancement of experimental of synaptic AMPA receptors is tightly regulated and may serve as a technology, i.e., from the clearance method to molecular biology. Molec- mechanism for information storage. Recent studies show that stargazin, ular biological studies have identified a wide variety of channel and the mutated protein in the ataxic and epileptic mouse stargazer, is neces- transporter proteins that exist in renal epithelial cells. Coupled with hu- sary for the expression of surface AMPA receptors in cerebellar granule man genetic studies it has been shown that mutations of the genes encod- cells. Stargazin is a small tetraspanning membrae protein and is a mem- ing renal membrane transport proteins (channels and transporters) cause ber of a family of proteins referred to as transmembrane AMPAR regu- human hereditary diseases such as water and solute loosing or retaining latory proteins (TARPs). These proteins are differentially expressed diseases. At present nearly all genes responsible for relatively popular throughout the brain. TARPs control AMPA receptor trafficking through human hereditary solute and water disorders have been determined and the endoplasmic reticulum and are important for the maturation and the current research interest is focusing on the mechanisms of regulation proper folding of AMPARs. They are necessary for the delivery of of the membrane transport proteins at molecular level. For this aim the AMPA receptors to the cell surface, as well as to the synapse. Finally disease-causing mutations found in hereditary diseases have provided TARPs control the gating of synaptic receptors. The role of TARPs is valuable information, for example, possible phosphorylation sites and analogous to non-pore forming auxiliary subunits of voltage gated ion protein-protein interaction domains. In this lecture, I will present our channels. Thus TARPs provide the first example of auxiliary subunits of data on water channels (aquaporin) and chloride channels and their dis- ionotropic receptors. In this talk I will review the pivotal role that TARPs eases of the kidney, and will show how molecular and genetic studies play in the life history of an AMPA receptor. work together to open a new field of the transport protein research. L2 (3PB) L4 (1EB) The Actin Cytoskeleton and Synaptic Epegenetics and Regulation of Gene Plasticity Expression through Nuclear Hormone Matus, Andrew (Friedrich Miescher Institute, Basel, Swiss Receptors Confederation) Kurokawa, Riki (Division of Gene Structure and Function, Dendritic spines form the postsynaptic contact elements for most excita- Research Center for Genomic Medicine, Saitama Medical School) tory synapses in the central nervous system. Using time-lapse imaging Coactivator and corepressor (cofactors) are essential for the transcrip- of living neurons expressing proteins tagged with green fluorescent pro- tional regulation by nuclear receptor (NR). Coactivator possesses intrin- tein (GFP) we discovered that dendritic spines undergo rapid changes in sic histone acetylase (HAT) activity and corepressor is associated with shape thus identifying them as the major sites of morphological plastic- histone deacetylase (HDAC). These enzyme activities are indispensable ity in neuronal circuits of the brain. This motility is driven by dynamic for the function of these cofactors, and also for modulation of chromatin actin filaments and is differentially regulated by various subtypes of structures, or epigenetic regulation. Recently, we have shown that dys- postsynaptic glutamate receptors. Activation of AMPA receptors pro- regulation of the HAT activity causes human diseases such as Rubin- duces an immediate blockade of spine motility which is reversed as soon stein-Taybi Syndrome (RTS) and Huntington's disease.Our present as the stimulus is withdrawn. By contrast, blockade of spine motility via efforts are to identify the roles of HAT activity in eukaryotic transcrip- NMDA receptors requires 30 min to develop and persists for hours after tional regulation. Recently we have found an RNA-binding protein, TLS the stimulus is withdrawn. To understand the cellular mechanisms un- (translocated in liposarcoma)/FUS, which has potent inhibitory effect on derlying these effects we examined the influence of actin binding pro- the HAT activity of CREB-binding protein (CBP). This inhibitory mol- teins in dendritic spines. Profilin shows activity-dependent targeting to ecule has a regulatory role in a CREB-dependent transcription system. spine heads which depends on activation of NMDA receptors and is in- Unexpectedly, specific RNA sequences stimulate the inhibitory activity duced by electrical stimulation patterns associated with changes in syn- of TLS. Such RNA sequence exists at 94% of coding regions of human aptic strength such as LTP. Simulataneously actin dynamics are genome. The specific RNAs form guanine (G) quartet structures in a K+- suppressed and spine motility is blocked for several hours. Conversely, dependent manner. This structure enhanced binding of the RNAs to TLS. blocking profilin targeting to spines by expressing a peptide that inhibits Addition of the RNAs in HAT assay of CBP with TLS resulted in more its binding to VASP family proteins destabilizes spine morphology. To- efficient inhibition to the HAT activity. These results have demonstrated gether with data for other actin binding proteins to be presented, this sug- that the RNAs with G-quartet structure function as ligands for TLS and gests that several distinct receptor dependent mechanisms regulate the that the TLS-RNA complex is a potent repressor for the CREB-depen- dynamic state of the spine actin cytoskeleton and hence morphological dent transcriptional systems, suggesting that TLS mediates RNA-depen- plasticity at excitatory synapses. dent transcriptional repression. S2 J. Physiol. Sci., Vol. 56, Suppl., 2006 Lectures L5 (1EC) L7 (3EC) Genetically engineered rodents in brain Clonal identification of mulltipotent stem/ research progenitor cells in the developing liver and Yanagawa, Yuchio1,2 (1Grad. Sch. Med. Gunma Univ., pancreas using flowcytometric cell sorting Maebashi, Japan; 2SORST, JST, Kawaguchi, Japan) Taniguchi, Hideki1,2 (1Department of Regenerative Medicin, Certain types of neurons in the brain are difficult to study because they Graduate School of Medicine, Yokohama City University; 2Research cannot be easily identified by location or morphological criteria alone. Unit for Organ Regeneration, Center for Divelopmental Biology, One approach to identify such neurons is to label them with a reporter RIKEN) protein. GABAergic inhibitory neurons play an important role in the reg- Using flowcytometry combined with single-cell-based assays, we pro- ulation and stabilization of network activities, but they are primarily spectively identified hepatic stem cells with multilineage differentiation scattered throughout mammalian central nervous systems and thus can potential and self-renewing capability in the developing mouse liver. c- be hardly identified in live brain preparations. GABA is synthesized Met+ CD49f+/low CD29+ c-Kit- CD45- TER119- cells in fetal liver from glutamic acid by glutamate decarboxylase (GAD) and is accumu- could be clonally propagated in culture, where they continuously pro- lated into synaptic vesicles by vesicular GABA transporter (VGAT). duced hepatocytes and cholangiocytes as descendants while maintaining Two isozymes of GAD, GAD65 and GAD67 and VGAT are primarily primitive stem cells. When cells that expanded in vitro were transplanted expressed in GABAergic neurons. To facilitate the study of GABAergic into recipient animals, they morphologically and functionally differenti- neurons, we generated the GAD67-GFP mice using a gene targeting ated into hepatocytes and cholangiocytes, with reconstitution of hepatic method via homologous recombination in ES cells. EGFP fluorescence cord and bile duct structures. These data indicate that self-renewing mul- was specifically observed in the GABAergic neurons in the GAD67- tipotent stem cells are retained in midgestational developing liver. The GFP mice. The GAD67-GFP mice have helped to elucidate the anatom- pancreas also contains a population of pancreatic stem cells that generate ical profile of GABA neuronal network and its electrophysiological ac- endocrine, exocrine, and ductal cells during development, neogenesis, tivity as well as the development of GABAergic neurons. In addition, we and regeneration. By combining flowcytometry and clonal analysis, we generated bacterial artificial chromosome transgenic rats, which specif- show here that stem/progenitor
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