{"created":"2023-06-20T13:21:07.207742+00:00","id":1218,"links":{},"metadata":{"_buckets":{"deposit":"be6ea3b1-acf7-4545-b666-a8498fe474da"},"_deposit":{"created_by":1,"id":"1218","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"1218"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00001218","sets":["2:431:23"]},"author_link":["0","0","0"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"田村, 洋"}],"nameIdentifiers":[{}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"タムラ, ヒロシ"}],"nameIdentifiers":[{}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2006-03-24"}]},"item_1_degree_grantor_5":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"総合研究大学院大学"}]}]},"item_1_degree_name_6":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（理学）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":"　　Protein tyrosine phosphatase receptor type Z(Ptprz) is receptor-type PTP that is<br />expressed predominantly in the CNS as a chondroitin sulfate proteoglycan. Although it<br />is expressed in neurons and astrocytes throughout development and adulthood,<br /> <i>Ptprz</i>-deficient (<i>Ptprz</i><sup>-/-</sup>) mice show no obvious anatomical abnormalities in the brain.<br />However, the recent study revealed that adult <i>Ptprz</i><sup>-/-</sup> mice exhibit functional<br /> impairments in the hippocampus, where Ptprz is highlyl expressed. 　Adult Pfprz-/- mice<br />show memory deficits in the Morris water maze, and demonstrate enhanced hippocampal<br />LTP in the CA1 region, which is cancelled out by inhibiting ROCK, a major downstream<br />effector of Rho GTPase. In addition, pl90 RhoGAP, a GTPase-activating protein<br />(GAP) which potently inhibits RhoGTPase, has been identified as a possible substrate of<br />Ptprz. These findings suggest that the Rho-ROCK pathway is impaired in <i>Ptprz</i>-/- mice, <br />however, little is known about the details of the intracellular signal transduction<br /> mechanism of Ptprz.<br />　　　This thesis investigates the possibility that Ptprz is involved in the molecular<br />mechanisms of memory formation through controlling the activity of p190 RhoGAP<br />using <i>Ptprz</i>-deficient (Ptprz<sup>-/-</sup>) mice. First, the hippocampus-dependent learning ability<br />of Ptprz<sup>-/-</sup> mice is analyzed by fear conditioning, which requires the association of an<br />environment with an aversive electric stimulus.　Second, to elucidate the molecular<br />mechanism by which Ptprz controls p190 RhoGAP activity, the site of dephosphorylation<br /> of p190 RhoGAP by Ptprz is identified, and the effect of phosphorylation at this site on<br />p190 RhoGAP activity is examined by a Rho activity assay.　Finally, the<br /> phosphorylation of p190 RhoGAP in the hippocampus, especially at the site of<br /> dephosphorylation by Ptprz, is compared between wild-type and mutant mice after fear<br />conditioning.<br />　　　To examine the hippocampus-dependent learning ability of Ptprz<sup>-/-</sup> mice in fear<br />conditioning, <i>Ptprz-/-</i> mice were tasted in two forms of behavioral tasks: contextual fear<br />conditioning and cued fear conditioning.　Both are sensitive to amygdala lesions, but<br />only contextual fear conditioning is sensitive to hippocampal lesions. <i>Ptprz</i>-/-mice<br />demonstrated marked impairements selectively in contextual fear conditioning.<br />　　　To further define the specificity of behavioral deficits, <i>Ptprz</i>-/- mice were subjected to<br />the elevated plus maze test in which fear or anxiety-related behaviors are analyzed.　　The<br />maze had two enclosed arms with high walls and two open arms with low rims and was<br />elevated above the foor. As mice usually avoid the open arms of maze, the extent of<br />anxiety can be evaluated by the time spent in the open arms relative to the closed arms.<br /> In addition, the numbers of entries into the arms of the maze likely corresponds to<br />general motor activity or exploratory activity. There were no significant differences<br />between the genotypes.<br />　　　<i>Ptprz</i>-/- mice exhibited normal vocalizing responses to an incremental series of<br />electric foot shocks and unaltered freezing responses during the conditioning session and<br />cued fear conditioning test, indicating that pain perception or emotional expression is not<br />affected in <i>Ptprz-/-</i> mice. Thus, <i>Ptprz</i>-/- mice did not show sensory or emotional deficits,<br /> but exhibited selective impairments in hippocampus-dependent behavioral tasks.<br />　　Second, to elucidate the molecular mechanism by which Ptprz controls p190<br /> RhoGAP activity, the site of dephosphorylation by Ptprz was determined by a mutation<br />study.　As Y1087 and Y1105 of p190 RhoGAP are reported to be the sites of tyrosine<br />phosphorylation on p190 RhoGAP, these sites were replaced with phenylalanine. Then,<br /> <i>in vitro</i> dephosphorylation assays with Ptprz were performed using wi1d-type p190<br />RhoGAP and its mutants. The Y1087F mutant as well as wild-type p190 RhoGAP was<br />efficiently dephosphorylated by the whole intracellular region of Ptprz while Y1105F and<br />Y1087/1105F mutants were not, indicating that Y1105 is the site of dephosphorylation by<br /> Ptprz.<br />　　　Next, the effect of the phospholylation at this site on p190 RhoGAP activity was<br />examined using wild-type p190 RhoGAP and Y1105F mutant.　GAP activity was<br />indirectly observed by the Rho activity assay, which detects the active fom of Rho.<br />Cotransfection of wild-type p190 RhoGAP and v-src in HEK293T cells led to an increase<br />in p190 RhoGAP tyrosine phosphorylation along with further inhibition of Rho,<br />indicating that increased tyrosine phosphorylation of p190 RhoGAP enhances its GAP<br />activity. 　On the other hand, Y1105F p190 RhoGAP, a mutant that can not be<br /> phosphorylated at Y1105, inhibited Rho to a comparable extent to wild-type p190<br />RhoGAP.　However, V-src did not increase the tyrosine phosphorylation of the Y1105F<br />mutant, and further inhibition of Rho was not observed, indicating that phosphorylation<br /> at Y1105 is critical for the regulation of p190 RhoGAP activity. Thus, tyrosine<br />phosphorylation at Y1105 positively controls the activity of p190 RhoGAP, which<br />indicates that p190 RhoGAP activity may be suppressed when p190 RhoGAP, is<br />dephosphorylated at Y1105 by Ptprz.<br />　　　Finally the phosphorylation of p190 RhoGAP in the hippocampus was compared<br />between wild-type and mutant mice after fear conditioning.　 To analyze the<br />phosphorylation of p190 RhoGAP especially at the site of dephosphorylation by Ptprz, a<br />phosphor-specific antibody (anti-p Y1105 p190 RhoGAP) directed against<br />Y1105 phosphorylated p190 RhoGAP) was generated by immunizing rabbits with the <br />tyrosine-phosphorylated peptide.　Then, hoppocampal homogenates were prepared from<br />animals after conditioning, and the effects of <i>Ptprz</i> knock-out on tyrosine<br />phosphorylation of hippocampal proteins were examined.<br/>　　　There were no significant differences in overall tyrosine phosphorylation patterns of<br />the hippocampal homogenates among the four groups. However, conditioned <i>Ptprz</i><sup>+/+</sup><br/>mice showed significantly reduced　p190 RhoGAP tyrosine phosphorylation compared<br />with sham-conditioned <i>Ptprz</i><sup>+/+</sup>mice.　In contrast, conditioned <i>Ptprz</i><sup>-/-</sup> mice exhibited<br />similar levels of tyrosine phosphorylation to sham-conditioned <i>Ptprz</i>mice.<br />Assessment by immunoblotting with anti-p Y1105 p190 RhoGAP antibody reveraled that<br />the level of p190 RhoGAP phosphorylated at Y1105 was significantly lower in<br />conditioned <i>Ptprz</i><sup>+/+</sup>mice than in sham-conditioned <i>Ptprz</i><sup>+/+</sup>mice, and almost identical<br />between conditioned and sham-conditioned <i>Ptprz</i><sup>+/+</sup>mice. These results can be<br />explained as indicating that only wild-type mice show dephosphorylation of p190<br />RhoGAP after fear conditioning, suggesting that p190 RhoGAP is dephosphorylated by<br />Ptprz after fear memory formation.<br />　　　Moreover, the phosphorylation of p190 RhoGAP at Y1105 in the hippocampus was<br />examined by immunohistochemistry using anti-p Y1105 p190 RhoGAP antibody. A<br />comparable level of p190 RhoGAP immunolabeling was confirmed among the four<br />groups by staining with anti-p190 RhoGAP antibody.　However, only sham-conditioned<br /> <i>Ptprz</i><sup>+/+</sup>mice showed significantly enhanced staining with anti-pY1105 p190 RhoGAP<br />antibody.　The immunostaining in sham-conditioned <i>Ptprz</i><sup>+/+</sup> mice was observed in the<br />stratum oriens as well as the stratum radiatum, in which Ptprz is prominently distributed.<br />These results indicate that dephosphorylation of p190 RhoGAP by Ptprz is involved in<br />fear memory formation.<br />　　　The present study demonstrated that the phosphorylation of p190 RhoGAP was<br />aberrantly regulated in <i>Ptprz</i><sup>-/-</sup> mice after fear conditioning.　 The level of <br />phosphorylation at Y1105 was decreased in conditioned <i>Ptprz</i><sup>+/+</sup>mice compared with<br />sham-conditioned <i>Ptprz</i><sup>+/+</sup>mice whereas no change was observed in <i>Ptprz</i><sup>-/-</sup>mice.<br />These results indicate that p190 RhoGAP activity is suppressed and consequently Rho<br />GTPase is activated after fear conditioning in wild-type mice, however,p190 RhoGAP<br />activity is maintained and subsequent Rho GTPase activation after learning does not<br />occur in mutant mice. The lack of Rho GTPase activity after learning is thus likely<br />responsible for the learning defects in Prprz-deficient mice, which is consistent with a<br />previous study in which post-training infusion of Y-27632 into the hippocampus impaired<br />spatial memory.<br />　　　In conclusion, Y1105 is the site of p190 RhoGAP dephosphorylation by Ptprz.<br />Ptprz inhibits p190 RhoGAP through dephosphorylation at this site, and consequently<br />activates Rho GTPase.　This regulation of p190 RhoGAP by Ptprz plays crucial roles in<br />the molecular mechanisms underlying hippocampus-dependent memory formation.","subitem_description_type":"Other"}]},"item_1_description_7":{"attribute_name":"学位記番号","attribute_value_mlt":[{"subitem_description":"総研大甲第978号","subitem_description_type":"Other"}]},"item_1_select_14":{"attribute_name":"所蔵","attribute_value_mlt":[{"subitem_select_item":"有"}]},"item_1_select_8":{"attribute_name":"研究科","attribute_value_mlt":[{"subitem_select_item":"先導科学研究科"}]},"item_1_select_9":{"attribute_name":"専攻","attribute_value_mlt":[{"subitem_select_item":"21 生命体科学専攻"}]},"item_1_text_10":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_text_value":"2005"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"TAMURA, Hiroshi","creatorNameLang":"en"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲978_要旨.pdf","filesize":[{"value":"487.2 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"要旨・審査要旨","url":"https://ir.soken.ac.jp/record/1218/files/甲978_要旨.pdf"},"version_id":"b0d163a4-d6da-4839-99ac-a7430c9f2517"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"Protein tyrosine phosphatase receptor type Z is involved in the molecular mechanisms of fear memory formation through regulating Y1105 phosphorylation of p190 RhoGAP","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Protein tyrosine phosphatase receptor type Z is involved in the molecular mechanisms of fear memory formation through regulating Y1105 phosphorylation of p190 RhoGAP"},{"subitem_title":"Protein tyrosine phosphatase receptor type Z is involved in the molecular mechanisms of fear memory formation through regulating Y1105 phosphorylation of p191 RhoGAP","subitem_title_language":"en"}]},"item_type_id":"1","owner":"1","path":["23"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-02-22"},"publish_date":"2010-02-22","publish_status":"0","recid":"1218","relation_version_is_last":true,"title":["Protein tyrosine phosphatase receptor type Z is involved in the molecular mechanisms of fear memory formation through regulating Y1105 phosphorylation of p190 RhoGAP"],"weko_creator_id":"1","weko_shared_id":-1},"updated":"2023-06-20T16:06:58.418347+00:00"}