{"_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": ["23"]}, "author_link": ["0", "0", "0"], "item_1_biblio_info_21": {"attribute_name": "書誌情報（ソート用）", "attribute_value_mlt": [{"bibliographicIssueDates": {"bibliographicIssueDate": "2006-03-24", "bibliographicIssueDateType": "Issued"}, "bibliographic_titles": [{}]}]}, "item_1_creator_2": {"attribute_name": "著者名", "attribute_type": "creator", "attribute_value_mlt": [{"creatorNames": [{"creatorName": "田村, 洋"}], "nameIdentifiers": [{"nameIdentifier": "0", "nameIdentifierScheme": "WEKO"}]}]}, "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": [{"nameIdentifier": "0", "nameIdentifierScheme": "WEKO"}]}]}, "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_1": {"attribute_name": "ID", "attribute_value_mlt": [{"subitem_description": "2006065", "subitem_description_type": "Other"}]}, "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\u003cbr /\u003eexpressed predominantly in the CNS as a chondroitin sulfate proteoglycan. Although it\u003cbr /\u003eis expressed in neurons and astrocytes throughout development and adulthood,\u003cbr /\u003e \u003ci\u003ePtprz\u003c/i\u003e-deficient (\u003ci\u003ePtprz\u003c/i\u003e\u003csup\u003e-/-\u003c/sup\u003e) mice show no obvious anatomical abnormalities in the brain.\u003cbr /\u003eHowever, the recent study revealed that adult \u003ci\u003ePtprz\u003c/i\u003e\u003csup\u003e-/-\u003c/sup\u003e mice exhibit functional\u003cbr /\u003e impairments in the hippocampus, where Ptprz is highlyl expressed. 　Adult Pfprz-/- mice\u003cbr /\u003eshow memory deficits in the Morris water maze, and demonstrate enhanced hippocampal\u003cbr /\u003eLTP in the CA1 region, which is cancelled out by inhibiting ROCK, a major downstream\u003cbr /\u003eeffector of Rho GTPase. In addition, pl90 RhoGAP, a GTPase-activating protein\u003cbr /\u003e(GAP) which potently inhibits RhoGTPase, has been identified as a possible substrate of\u003cbr /\u003ePtprz. These findings suggest that the Rho-ROCK pathway is impaired in \u003ci\u003ePtprz\u003c/i\u003e-/- mice, \u003cbr /\u003ehowever, little is known about the details of the intracellular signal transduction\u003cbr /\u003e mechanism of Ptprz.\u003cbr /\u003e　　　This thesis investigates the possibility that Ptprz is involved in the molecular\u003cbr /\u003emechanisms of memory formation through controlling the activity of p190 RhoGAP\u003cbr /\u003eusing \u003ci\u003ePtprz\u003c/i\u003e-deficient (Ptprz\u003csup\u003e-/-\u003c/sup\u003e) mice. First, the hippocampus-dependent learning ability\u003cbr /\u003eof Ptprz\u003csup\u003e-/-\u003c/sup\u003e mice is analyzed by fear conditioning, which requires the association of an\u003cbr /\u003eenvironment with an aversive electric stimulus.　Second, to elucidate the molecular\u003cbr /\u003emechanism by which Ptprz controls p190 RhoGAP activity, the site of dephosphorylation\u003cbr /\u003e of p190 RhoGAP by Ptprz is identified, and the effect of phosphorylation at this site on\u003cbr /\u003ep190 RhoGAP activity is examined by a Rho activity assay.　Finally, the\u003cbr /\u003e phosphorylation of p190 RhoGAP in the hippocampus, especially at the site of\u003cbr /\u003e dephosphorylation by Ptprz, is compared between wild-type and mutant mice after fear\u003cbr /\u003econditioning.\u003cbr /\u003e　　　To examine the hippocampus-dependent learning ability of Ptprz\u003csup\u003e-/-\u003c/sup\u003e mice in fear\u003cbr /\u003econditioning, \u003ci\u003ePtprz-/-\u003c/i\u003e mice were tasted in two forms of behavioral tasks: contextual fear\u003cbr /\u003econditioning and cued fear conditioning.　Both are sensitive to amygdala lesions, but\u003cbr /\u003eonly contextual fear conditioning is sensitive to hippocampal lesions. \u003ci\u003ePtprz\u003c/i\u003e-/-mice\u003cbr /\u003edemonstrated marked impairements selectively in contextual fear conditioning.\u003cbr /\u003e　　　To further define the specificity of behavioral deficits, \u003ci\u003ePtprz\u003c/i\u003e-/- mice were subjected to\u003cbr /\u003ethe elevated plus maze test in which fear or anxiety-related behaviors are analyzed.　　The\u003cbr /\u003emaze had two enclosed arms with high walls and two open arms with low rims and was\u003cbr /\u003eelevated above the foor. As mice usually avoid the open arms of maze, the extent of\u003cbr /\u003eanxiety can be evaluated by the time spent in the open arms relative to the closed arms.\u003cbr /\u003e In addition, the numbers of entries into the arms of the maze likely corresponds to\u003cbr /\u003egeneral motor activity or exploratory activity. There were no significant differences\u003cbr /\u003ebetween the genotypes.\u003cbr /\u003e　　　\u003ci\u003ePtprz\u003c/i\u003e-/- mice exhibited normal vocalizing responses to an incremental series of\u003cbr /\u003eelectric foot shocks and unaltered freezing responses during the conditioning session and\u003cbr /\u003ecued fear conditioning test, indicating that pain perception or emotional expression is not\u003cbr /\u003eaffected in \u003ci\u003ePtprz-/-\u003c/i\u003e mice. Thus, \u003ci\u003ePtprz\u003c/i\u003e-/- mice did not show sensory or emotional deficits,\u003cbr /\u003e but exhibited selective impairments in hippocampus-dependent behavioral tasks.\u003cbr /\u003e　　Second, to elucidate the molecular mechanism by which Ptprz controls p190\u003cbr /\u003e RhoGAP activity, the site of dephosphorylation by Ptprz was determined by a mutation\u003cbr /\u003estudy.　As Y1087 and Y1105 of p190 RhoGAP are reported to be the sites of tyrosine\u003cbr /\u003ephosphorylation on p190 RhoGAP, these sites were replaced with phenylalanine. Then,\u003cbr /\u003e \u003ci\u003ein vitro\u003c/i\u003e dephosphorylation assays with Ptprz were performed using wi1d-type p190\u003cbr /\u003eRhoGAP and its mutants. The Y1087F mutant as well as wild-type p190 RhoGAP was\u003cbr /\u003eefficiently dephosphorylated by the whole intracellular region of Ptprz while Y1105F and\u003cbr /\u003eY1087/1105F mutants were not, indicating that Y1105 is the site of dephosphorylation by\u003cbr /\u003e Ptprz.\u003cbr /\u003e　　　Next, the effect of the phospholylation at this site on p190 RhoGAP activity was\u003cbr /\u003eexamined using wild-type p190 RhoGAP and Y1105F mutant.　GAP activity was\u003cbr /\u003eindirectly observed by the Rho activity assay, which detects the active fom of Rho.\u003cbr /\u003eCotransfection of wild-type p190 RhoGAP and v-src in HEK293T cells led to an increase\u003cbr /\u003ein p190 RhoGAP tyrosine phosphorylation along with further inhibition of Rho,\u003cbr /\u003eindicating that increased tyrosine phosphorylation of p190 RhoGAP enhances its GAP\u003cbr /\u003eactivity. 　On the other hand, Y1105F p190 RhoGAP, a mutant that can not be\u003cbr /\u003e phosphorylated at Y1105, inhibited Rho to a comparable extent to wild-type p190\u003cbr /\u003eRhoGAP.　However, V-src did not increase the tyrosine phosphorylation of the Y1105F\u003cbr /\u003emutant, and further inhibition of Rho was not observed, indicating that phosphorylation\u003cbr /\u003e at Y1105 is critical for the regulation of p190 RhoGAP activity. Thus, tyrosine\u003cbr /\u003ephosphorylation at Y1105 positively controls the activity of p190 RhoGAP, which\u003cbr /\u003eindicates that p190 RhoGAP activity may be suppressed when p190 RhoGAP, is\u003cbr /\u003edephosphorylated at Y1105 by Ptprz.\u003cbr /\u003e　　　Finally the phosphorylation of p190 RhoGAP in the hippocampus was compared\u003cbr /\u003ebetween wild-type and mutant mice after fear conditioning.　 To analyze the\u003cbr /\u003ephosphorylation of p190 RhoGAP especially at the site of dephosphorylation by Ptprz, a\u003cbr /\u003ephosphor-specific antibody (anti-p Y1105 p190 RhoGAP) directed against\u003cbr /\u003eY1105 phosphorylated p190 RhoGAP) was generated by immunizing rabbits with the \u003cbr /\u003etyrosine-phosphorylated peptide.　Then, hoppocampal homogenates were prepared from\u003cbr /\u003eanimals after conditioning, and the effects of \u003ci\u003ePtprz\u003c/i\u003e knock-out on tyrosine\u003cbr /\u003ephosphorylation of hippocampal proteins were examined.\u003cbr/\u003e　　　There were no significant differences in overall tyrosine phosphorylation patterns of\u003cbr /\u003ethe hippocampal homogenates among the four groups. However, conditioned \u003ci\u003ePtprz\u003c/i\u003e\u003csup\u003e+/+\u003c/sup\u003e\u003cbr/\u003emice showed significantly reduced　p190 RhoGAP tyrosine phosphorylation compared\u003cbr /\u003ewith sham-conditioned \u003ci\u003ePtprz\u003c/i\u003e\u003csup\u003e+/+\u003c/sup\u003emice.　In contrast, conditioned \u003ci\u003ePtprz\u003c/i\u003e\u003csup\u003e-/-\u003c/sup\u003e mice exhibited\u003cbr /\u003esimilar levels of tyrosine phosphorylation to sham-conditioned \u003ci\u003ePtprz\u003c/i\u003emice.\u003cbr /\u003eAssessment by immunoblotting with anti-p Y1105 p190 RhoGAP antibody reveraled that\u003cbr /\u003ethe level of p190 RhoGAP phosphorylated at Y1105 was significantly lower in\u003cbr /\u003econditioned \u003ci\u003ePtprz\u003c/i\u003e\u003csup\u003e+/+\u003c/sup\u003emice than in sham-conditioned \u003ci\u003ePtprz\u003c/i\u003e\u003csup\u003e+/+\u003c/sup\u003emice, and almost identical\u003cbr /\u003ebetween conditioned and sham-conditioned \u003ci\u003ePtprz\u003c/i\u003e\u003csup\u003e+/+\u003c/sup\u003emice. These results can be\u003cbr /\u003eexplained as indicating that only wild-type mice show dephosphorylation of p190\u003cbr /\u003eRhoGAP after fear conditioning, suggesting that p190 RhoGAP is dephosphorylated by\u003cbr /\u003ePtprz after fear memory formation.\u003cbr /\u003e　　　Moreover, the phosphorylation of p190 RhoGAP at Y1105 in the hippocampus was\u003cbr /\u003eexamined by immunohistochemistry using anti-p Y1105 p190 RhoGAP antibody. A\u003cbr /\u003ecomparable level of p190 RhoGAP immunolabeling was confirmed among the four\u003cbr /\u003egroups by staining with anti-p190 RhoGAP antibody.　However, only sham-conditioned\u003cbr /\u003e \u003ci\u003ePtprz\u003c/i\u003e\u003csup\u003e+/+\u003c/sup\u003emice showed significantly enhanced staining with anti-pY1105 p190 RhoGAP\u003cbr /\u003eantibody.　The immunostaining in sham-conditioned \u003ci\u003ePtprz\u003c/i\u003e\u003csup\u003e+/+\u003c/sup\u003e mice was observed in the\u003cbr /\u003estratum oriens as well as the stratum radiatum, in which Ptprz is prominently distributed.\u003cbr /\u003eThese results indicate that dephosphorylation of p190 RhoGAP by Ptprz is involved in\u003cbr /\u003efear memory formation.\u003cbr /\u003e　　　The present study demonstrated that the phosphorylation of p190 RhoGAP was\u003cbr /\u003eaberrantly regulated in \u003ci\u003ePtprz\u003c/i\u003e\u003csup\u003e-/-\u003c/sup\u003e mice after fear conditioning.　 The level of \u003cbr /\u003ephosphorylation at Y1105 was decreased in conditioned \u003ci\u003ePtprz\u003c/i\u003e\u003csup\u003e+/+\u003c/sup\u003emice compared with\u003cbr /\u003esham-conditioned \u003ci\u003ePtprz\u003c/i\u003e\u003csup\u003e+/+\u003c/sup\u003emice whereas no change was observed in \u003ci\u003ePtprz\u003c/i\u003e\u003csup\u003e-/-\u003c/sup\u003emice.\u003cbr /\u003eThese results indicate that p190 RhoGAP activity is suppressed and consequently Rho\u003cbr /\u003eGTPase is activated after fear conditioning in wild-type mice, however,p190 RhoGAP\u003cbr /\u003eactivity is maintained and subsequent Rho GTPase activation after learning does not\u003cbr /\u003eoccur in mutant mice. The lack of Rho GTPase activity after learning is thus likely\u003cbr /\u003eresponsible for the learning defects in Prprz-deficient mice, which is consistent with a\u003cbr /\u003eprevious study in which post-training infusion of Y-27632 into the hippocampus impaired\u003cbr /\u003espatial memory.\u003cbr /\u003e　　　In conclusion, Y1105 is the site of p190 RhoGAP dephosphorylation by Ptprz.\u003cbr /\u003ePtprz inhibits p190 RhoGAP through dephosphorylation at this site, and consequently\u003cbr /\u003eactivates Rho GTPase.　This regulation of p190 RhoGAP by Ptprz plays crucial roles in\u003cbr /\u003ethe 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": [{"nameIdentifier": "0", "nameIdentifierScheme": "WEKO"}]}]}, "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", "download_preview_message": "", "file_order": 0, "filename": "甲978_要旨.pdf", "filesize": [{"value": "487.2 kB"}], "format": "application/pdf", "future_date_message": "", "is_thumbnail": false, "licensetype": "license_11", "mimetype": "application/pdf", "size": 487200.0, "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"], "permalink_uri": "https://ir.soken.ac.jp/records/1218", "pubdate": {"attribute_name": "公開日", "attribute_value": "2010-02-22"}, "publish_date": "2010-02-22", "publish_status": "0", "recid": "1218", "relation": {}, "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_shared_id": -1}