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内容記述 |
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. |
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