{"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":[{"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_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":"  Protein tyrosine phosphatase receptor type Z(Ptprz) is receptor-type PTP that is
expressed predominantly in the CNS as a chondroitin sulfate proteoglycan. Although it
is expressed in neurons and astrocytes throughout development and adulthood,
Ptprz-deficient (Ptprz-/-) mice show no obvious anatomical abnormalities in the brain.
However, the recent study revealed that adult Ptprz-/- mice exhibit functional
impairments in the hippocampus, where Ptprz is highlyl expressed.  Adult Pfprz-/- mice
show memory deficits in the Morris water maze, and demonstrate enhanced hippocampal
LTP in the CA1 region, which is cancelled out by inhibiting ROCK, a major downstream
effector of Rho GTPase. In addition, pl90 RhoGAP, a GTPase-activating protein
(GAP) which potently inhibits RhoGTPase, has been identified as a possible substrate of
Ptprz. These findings suggest that the Rho-ROCK pathway is impaired in Ptprz-/- mice,
however, little is known about the details of the intracellular signal transduction
mechanism of Ptprz.
   This thesis investigates the possibility that Ptprz is involved in the molecular
mechanisms of memory formation through controlling the activity of p190 RhoGAP
using Ptprz-deficient (Ptprz-/-) mice. First, the hippocampus-dependent learning ability
of Ptprz-/- mice is analyzed by fear conditioning, which requires the association of an
environment with an aversive electric stimulus. Second, to elucidate the molecular
mechanism by which Ptprz controls p190 RhoGAP activity, the site of dephosphorylation
of p190 RhoGAP by Ptprz is identified, and the effect of phosphorylation at this site on
p190 RhoGAP activity is examined by a Rho activity assay. Finally, the
phosphorylation of p190 RhoGAP in the hippocampus, especially at the site of
dephosphorylation by Ptprz, is compared between wild-type and mutant mice after fear
conditioning.
   To examine the hippocampus-dependent learning ability of Ptprz-/- mice in fear
conditioning, Ptprz-/- mice were tasted in two forms of behavioral tasks: contextual fear
conditioning and cued fear conditioning. Both are sensitive to amygdala lesions, but
only contextual fear conditioning is sensitive to hippocampal lesions. Ptprz-/-mice
demonstrated marked impairements selectively in contextual fear conditioning.
   To further define the specificity of behavioral deficits, Ptprz-/- mice were subjected to
the elevated plus maze test in which fear or anxiety-related behaviors are analyzed.  The
maze had two enclosed arms with high walls and two open arms with low rims and was
elevated above the foor. As mice usually avoid the open arms of maze, the extent of
anxiety can be evaluated by the time spent in the open arms relative to the closed arms.
In addition, the numbers of entries into the arms of the maze likely corresponds to
general motor activity or exploratory activity. There were no significant differences
between the genotypes.
   Ptprz-/- mice exhibited normal vocalizing responses to an incremental series of
electric foot shocks and unaltered freezing responses during the conditioning session and
cued fear conditioning test, indicating that pain perception or emotional expression is not
affected in Ptprz-/- mice. Thus, Ptprz-/- mice did not show sensory or emotional deficits,
but exhibited selective impairments in hippocampus-dependent behavioral tasks.
  Second, to elucidate the molecular mechanism by which Ptprz controls p190
RhoGAP activity, the site of dephosphorylation by Ptprz was determined by a mutation
study. As Y1087 and Y1105 of p190 RhoGAP are reported to be the sites of tyrosine
phosphorylation on p190 RhoGAP, these sites were replaced with phenylalanine. Then,
in vitro dephosphorylation assays with Ptprz were performed using wi1d-type p190
RhoGAP and its mutants. The Y1087F mutant as well as wild-type p190 RhoGAP was
efficiently dephosphorylated by the whole intracellular region of Ptprz while Y1105F and
Y1087/1105F mutants were not, indicating that Y1105 is the site of dephosphorylation by
Ptprz.
   Next, the effect of the phospholylation at this site on p190 RhoGAP activity was
examined using wild-type p190 RhoGAP and Y1105F mutant. GAP activity was
indirectly observed by the Rho activity assay, which detects the active fom of Rho.
Cotransfection of wild-type p190 RhoGAP and v-src in HEK293T cells led to an increase
in p190 RhoGAP tyrosine phosphorylation along with further inhibition of Rho,
indicating that increased tyrosine phosphorylation of p190 RhoGAP enhances its GAP
activity.  On the other hand, Y1105F p190 RhoGAP, a mutant that can not be
phosphorylated at Y1105, inhibited Rho to a comparable extent to wild-type p190
RhoGAP. However, V-src did not increase the tyrosine phosphorylation of the Y1105F
mutant, and further inhibition of Rho was not observed, indicating that phosphorylation
at Y1105 is critical for the regulation of p190 RhoGAP activity. Thus, tyrosine
phosphorylation at Y1105 positively controls the activity of p190 RhoGAP, which
indicates that p190 RhoGAP activity may be suppressed when p190 RhoGAP, is
dephosphorylated at Y1105 by Ptprz.
   Finally the phosphorylation of p190 RhoGAP in the hippocampus was compared
between wild-type and mutant mice after fear conditioning.  To analyze the
phosphorylation of p190 RhoGAP especially at the site of dephosphorylation by Ptprz, a
phosphor-specific antibody (anti-p Y1105 p190 RhoGAP) directed against
Y1105 phosphorylated p190 RhoGAP) was generated by immunizing rabbits with the
tyrosine-phosphorylated peptide. Then, hoppocampal homogenates were prepared from
animals after conditioning, and the effects of Ptprz knock-out on tyrosine
phosphorylation of hippocampal proteins were examined.
   There were no significant differences in overall tyrosine phosphorylation patterns of
the hippocampal homogenates among the four groups. However, conditioned Ptprz+/+
mice showed significantly reduced p190 RhoGAP tyrosine phosphorylation compared
with sham-conditioned Ptprz+/+mice. In contrast, conditioned Ptprz-/- mice exhibited
similar levels of tyrosine phosphorylation to sham-conditioned Ptprzmice.
Assessment by immunoblotting with anti-p Y1105 p190 RhoGAP antibody reveraled that
the level of p190 RhoGAP phosphorylated at Y1105 was significantly lower in
conditioned Ptprz+/+mice than in sham-conditioned Ptprz+/+mice, and almost identical
between conditioned and sham-conditioned Ptprz+/+mice. These results can be
explained as indicating that only wild-type mice show dephosphorylation of p190
RhoGAP after fear conditioning, suggesting that p190 RhoGAP is dephosphorylated by
Ptprz after fear memory formation.
   Moreover, the phosphorylation of p190 RhoGAP at Y1105 in the hippocampus was
examined by immunohistochemistry using anti-p Y1105 p190 RhoGAP antibody. A
comparable level of p190 RhoGAP immunolabeling was confirmed among the four
groups by staining with anti-p190 RhoGAP antibody. However, only sham-conditioned
Ptprz+/+mice showed significantly enhanced staining with anti-pY1105 p190 RhoGAP
antibody. The immunostaining in sham-conditioned Ptprz+/+ mice was observed in the
stratum oriens as well as the stratum radiatum, in which Ptprz is prominently distributed.
These results indicate that dephosphorylation of p190 RhoGAP by Ptprz is involved in
fear memory formation.
   The present study demonstrated that the phosphorylation of p190 RhoGAP was
aberrantly regulated in Ptprz-/- mice after fear conditioning.  The level of
phosphorylation at Y1105 was decreased in conditioned Ptprz+/+mice compared with
sham-conditioned Ptprz+/+mice whereas no change was observed in Ptprz-/-mice.
These results indicate that p190 RhoGAP activity is suppressed and consequently Rho
GTPase is activated after fear conditioning in wild-type mice, however,p190 RhoGAP
activity is maintained and subsequent Rho GTPase activation after learning does not
occur in mutant mice. The lack of Rho GTPase activity after learning is thus likely
responsible for the learning defects in Prprz-deficient mice, which is consistent with a
previous study in which post-training infusion of Y-27632 into the hippocampus impaired
spatial memory.
   In conclusion, Y1105 is the site of p190 RhoGAP dephosphorylation by Ptprz.
Ptprz inhibits p190 RhoGAP through dephosphorylation at this site, and consequently
activates Rho GTPase. This regulation of p190 RhoGAP by Ptprz plays crucial roles in
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":[{"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","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"}