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内容記述 |
Fyn, a non-receptor tyrosine kinase, is expressed in lymphocytes and neural system. In the brain, Fyn plays a variety of roles in neural development, and in synaptic plasticity after neural maturation. This information comes largely from knockout approach. Care should be paid, however, in interpreting the results of fyn-knockout mice; the morphological abnormalities, which results from the absence of Fyn in the developmental period, may have influences in the mature neural system. To dissociate Fyn's function in the adult brain from that in the neural developmental period, Kojima generated fyn-transgenic mice, in which transgene expression started postnatally. Aiming at searching the functions of Fyn tyrosine kinase in the mature brains, he used the advantage of fyn-trangenic mice; the logic is that the phenotypes in fyn-transgenic mice would reflect the roles of Fyn in the adult brain.<br /> Tyrosine phosphorylation of proteins in the adult brains was investigated by immunoblotting method using anti-phosphotyrosine antibody. Five proteins were hyper-phosphorylated in fyn-transgenic mice's brains, and hypo-phosphorylated in fyn-deficient mice's brains, suggesting that these five proteins are the substrates of Fyn in the adult brains. Subcellular fractionation revealed that four of the five proteins, designated as PY180, PY120, PY110 and PY100 by their electromobility, and Fyn localized to membrane fraction. He identified PY180, by immunoprecipitation and immunoblotting method, to be the modulatory subunit 2B of the NMDA receptor (NR2B).<br /> Two behavioral phenotypes of fyn-transgenic mice were found. Epileptic phenotype was first noticed by occasional spontaneous seizures characterized by running and bouncing fits. Two seizure-inducing experiments, infusion of a convulsannt drug pentylenetetrazol, and electrical kindling protocol, revealed<br />acceletared seizire development in fyn-transgenic mice. The administration of NMDA receptor antagonist MK-801 completely suppressed the accelerated development of clonic seizure (the significant difference without the drug, p<0.0001, disappeared in the presence of the drug, p>0.1, revealed by two-way ANOVA with a repeated measure as a day-series). These results suggest the involvement of the tyrosine phosphorylation of the NMDA receptors in developing clonic seizure.<br /> The other behavioral phenotype, akinesia, was also analyzed. While the count of the spontaneous locomotion of fyn-transgenic mice was significantly lower than that of wild-type control mice (t30=2.89, p=0.0071 with two-tailed t test), circadian rhythm of the locomotor activity was not different from that of control mice. Furthermore, general behavior was not different between two groups. The administration of NMDA receptor antagonist MK-801 dose-dependency recovered the hypokinesia in fyn-transgenic mice; i.e. two-way ANOVA revealed significant effects in the drug dose (F2.49=13.48, p<0.0001) and in the interaction between the drug dose and the genetic group (F2.49=6.26, p=0.0038), but not in the genetic group (F1.49=3. 45, p=0.069), showing that the locomotor-enhancing effect of the drug was stronger in fyn-transgenic mice than in wild-type control mice. These results suggest the involvement of the tyrosine phosphorylation of NMDA receptors in the gain control of total locomotor activity, but not in the initiation of specific behavior.<br /> Since the activated NMDA receptors, at least in part, mediated both epileptogenesis and hypokinesia in fyn-transgenic mice, he inferred close relation between Fyn tyrosine kinase and NMDA receptors, and thus speculated fyn-transgenic mice possessed other phenotypes, which were mediated through the NMDA receptor functions. He, therefore, examined learning and memory abilities thought to depend on the NMDA receptor activity. Contrary to the prediction, tests of three types of learning and memory, passive avoidance learning, spatial learning and odor discrimination learning, revealed no differences in the performance between wild type control mice and fyn-transgenic mice. These results suggest that the tyrosine phosphorylation of the NMDA receptors does not enhance the learning and memory abilities. Therefore, the tyrosine phosphorylation of NMDA receptors enhances the functions of NMDA receptors in some occasion, but not in another occasion.<br /> In conclusion, he found two phenotypes in adult fyn-transgenic mice; epileptogenesis and akinesia. Both phenotypes were mediated, at least in part, by the activated NMDA receptors. Consistent with these results, he found enhanced tyrosine phosphorylation of the subunit 2B of the NMDA receptors. These results suggest two points. First, some functions of Fyn are mediated through the tyrosine phosphorylation of the NMDA receptors. Second, the tyrosine phosphorylation of the NR2B is involved in expressing the NMDA receptor functions in some occasion in the brain. |
要旨・審査要旨 / Abstract, Screening Result (318.7 kB)
