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Conditional Phgdh deletion results in reduced D,L-serine levels and alters monoamine metabolism in the postnatal brain
Abstracts / Neuroscience Research 68S (2010) e109–e222
age-dependent decrease, as follows; 215.3 ± 28.1 ng/ml in 18–19 y.o. (n = 5), 183.4 ± 6.8 ng/ml in 20–29 y.o. (n = 58), 161.9 ± 7.9 ng/ml in 30–39 y.o. (n = 44), 152.8 ± 8.3 ng/ml in 40–49 y.o. (n = 31), 154.2 ± 9.8 ng/ml in 50–59 y.o. (n = 25) and 108.5 ± 10.2 ng/ml over 60 y.o. (n = 9). On the other hand, we had an opportunity to measure WB 5-HT levels in depressive subjects (3 males and 4 females, age 29–46 years) with a treatment of selective serotonin reuptake inhibitors (SSRIs; Paxil, Luvox or Depromel). These subjects showed an extremely lower WB 5-HT level (14.1 ± 3.8 ng/ml), as compared to healthy subjects. This may be explained by the mechanism that SSRIs could inhibit 5-HT transporters located at blood-brain barrier as well as pre-synaptic terminals of 5-HT neurons. doi:10.1016/j.neures.2010.07.2094
P1-b20 Conditional Phgdh deletion results in reduced D,Lserine levels and alters monoamine metabolism in the postnatal brain Shozo Tomonaga 1 , Kayoko Esaki 2 , Akira Wada 2 , Satoko Yang 2 , Toshihisa Ohshima 3 , Mitsuhiro Sayano 2 , JungHoon Furuse 4 , Yoshio Hirabayashi 5 , Shigeki Furuya 2 1
Facul Agr, Kyushu Univ, Fukuoka 2 Facul Agr, Kyushu Univ, Fukuoka 3 Facul Agr, Kyushu Univ, Fukuoka 4 Facul Agr, Kyushu Univ, Fukuoka 5 RIKEN BSI, Wako The mammalian brain contains D-serine at high levels. The amino acid is synthesized from L-serine by serine racemase, and it functions as an obligatory coagonist of NMDA receptors. We previously revealed that conditional deletion of brain Phgdh, which catalyzes the first step of L-serine synthesis, resulted in reduced D, L-serine levels in the adult brain. In the present study, we found significant decreases in D- and L-serine contents in the postnatal brain of this conditional knockout mouse. Furthermore, metabolism of monoamines is altered simultaneously in particular regions in the postnatal brain. These results raise the possibility that the availability of L-serine may play a regulatory role in an interaction between glutamatergic and monoamine systems probably via D-serine during the postnatal developmental period. doi:10.1016/j.neures.2010.07.2095
e119
P1-b22 Phosphorylation of Cyclin-dependent kinase 5 at Tyrosine 15 Hiroyuki Kobayashi , Ko Sato, Tomohisa Hosokawa, Taro Saitou, Shin-Ichi Hisanaga Molecular Neurobiology, Department of Biological Sciences Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase activated by binding to brain specific regulatory subunit p35 or p39. Different from other active Cdks in proliferating cells (Cycling Cdks), which require the phosphorylation at Thr161 in the activation loop for activation, Cdk5 is activated only by binding to p35 or p39. Furthermore, in contrast to cycling Cdks, whose kinase activity is inhibited by phosphorylation at tyrosine 15 (Tyr15), Cdk5 is activated by phosphorylation at Tyr15 with Src family kinases or receptor type tyrosine kinases. Activation of Cdk5-p35 by Tyr15 phosphorylation is indicated to induce neurite retraction and spine formation. Thus, phosphorylation of Cdk5 at Tyr15 appears to play an important role in several neuronal activities. However, it is not known well how Tyr15 phosphorylation activates Cdk5. In this study, we have investigated phosphorylation of Cdk5 at Tyr15 using Src family kinases. We transfected Cdk5 and p35 together with Fyn into COS-7 cells and observed phosphorylation of Cdk5 using anti-phospho-Tyr15. However, we could not detect the reaction even though constitutive active Fyn was coexpressed. Unexpectedly, phosphorylation of Cdk5 at Tyr15 was found when Cdk5 alone was expressed in COS-7 cells with active Fyn. We suspected that active Cdk5-p35 inhibits Fyn kinase, and transfected a kinase negative Cdk5 mutant (D144N) with Fyn. Tyr15 phosphorylation was still diminished by coexpression with p35. We wondered the N-terminal region of p35 impairs Tyr15 phosphorylation sterically and coexpressed with p25, N-terminal truncation form of p25. But phosphorylation of Cdk5 was also inhibited by p25. These results suggest that Cdk5 is phosphorylated at Tyr15 only when it is a monomeric free form. These results claim the re-evaluation of Tyr15 phosphorylation on Cdk5 activation. doi:10.1016/j.neures.2010.07.2097
P1-b23 Transactivation of ErbB receptor by GnRH via CaM kinase II in cultured hypothalamic neurons Hideyuki Yamamoto 1 , Sayomi Higa-Nakamine 1 , Noriko Maeda 1 , Tomoko Yamamoto 1 , Seikichi Toku 1 , Masahiro Kawahara 2 1
P1-b21 Effect of bisphenol A exposure during gestation and lactation on amino acid metabolism in the rat brain of offspring Hirokazu Mizokawa Tomoyuki Kanamatsu
, George
Kokubu, Shinichi
Sakamoto,
Department of Environmental Engineering for Symbiosis, Soka university, Tokyo, Japan Exposure to bisphenol A (BPA) during the gestation and lactation periods alters the locomotor activity and learning ability of mature rat offspring. The influences of BPA are caused through estrogen receptor. Estrogen is involved in organogenesis period of brain. The purpose of this study was to elucidate the effect of BPA on amino acid metabolism in the rat brain. The BPA-exposure groups were treated with 0.1, 1.0, and 5.0 ppm BPA in drinking water, respectively, and control group was given tap water. All weaning rats were kept giving the tap water. In this study, male offspring rats between 18 and 24 weeks of age were used. The rats were killed with the microwave treatment under anesthesia 20 min after the injection of [1-13 C]glucose via the tail vein. Brain regions were dissected into frontal cortex, thalamus, striatum, hippocampus and brain stem according to the method of Glowinski and Iverson, then, the amino acid fraction was extracted from each tissue. The extracted fractions were analyzed by 13 C-NMR spectroscopy. The 13 C incorporation into GABA C2 from [1-13 C]glucose was significantly lower in the thalamus and higher in the striatum in 0.1 ppm BPA-exposed group compared to the control group. In other brain regions, there was no significant difference in the 13 C incorporation between four groups. In GABA concentration, there was no significant difference in the thalamus, and a significant increase was shown in the striatum. These results suggest that exposure to BPA during the gestation and lactation may affect GABA (inhibitory neurotransmitter) metabolism in the brain of offspring. An alteration of GABA metabolism may cause the behavioral abnormality in the rat offspring. doi:10.1016/j.neures.2010.07.2096
Dept Biochem, University of the Ryukyus, Nishihara, Japan 2 Dept of Analytical Chem, Kyusyu Univ. of Health and Welfare, Miyazaki, Japan Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic neurons (GnRH neurons). It has been reported that GnRH neurons have GnRH receptor and that the autocrine action of GnRH regulates the functions of GnRH neurons. We have reported that treatment of the immortalized GnRH neurons (GT1-7 cells) with GnRH activated MAP kinase via activation of CaM kinase II. In the present study, we found that activation of MAP kinase was inhibited both by AG1478, an inhibitor of ErbB receptor tyrosine kinase family, and by GM6001, an inhibitor of matrix metalloproteinases. In contrast, TAPI-2, an inhibitor of TACE had no effects on MAP kinase activation. Immunoblot analysis and sequence after RT-PCR indicated that ErbB4, but not epidermal growth factor receptor, was expressed in GT1-7 cells. These results suggested that the activation of CaM kinase II by GnRH induced the activation of MAP kinase via transactivation of ErbB4 in GT1-7 cells. doi:10.1016/j.neures.2010.07.2098
P1-b25 G protein Rho and Rho-kinase regulate the augmenting effect of 5HT on the voltage-dependent Ca2+ current
Noriyuki Watanabe 1 , Satoshi Kawasaki 1 , Shingo Kimura 1 , Misato Harata 1 , Reiko Fujita 2 , Kazuhiko Sasaki 1 1
Dept Physiol, Sch Med, Iwate Med Univ 2 Dept Chemistry, Ctr Lib Arts & Sci, Iwate Med Univ We previously reported that small G protein Rho is involved in the facilitation of the inward current responses to serotonin (5-HT) in the ganglion cells of Aplysia under voltage-clamp. To investigate other role of Rho in the receptor-induced responses in the neuron, we studied action of Rho on the 5-HT-induced facilitation of the voltage-dependent Ca2+ current using conventional two-electrode voltage clamp method in combination with intracellular injection of various enzymes and reagents. We measured a depolarization-induced outward current as indicative of voltage-dependent Ca2+ current to avoid the loss of physiological circumstance. Application of 5-
age-dependent decrease, as follows; 215.3 ± 28.1 ng/ml in 18–19 y.o. (n = 5), 183.4 ± 6.8 ng/ml in 20–29 y.o. (n = 58), 161.9 ± 7.9 ng/ml in 30–39 y.o. (n = 44), 152.8 ± 8.3 ng/ml in 40–49 y.o. (n = 31), 154.2 ± 9.8 ng/ml in 50–59 y.o. (n = 25) and 108.5 ± 10.2 ng/ml over 60 y.o. (n = 9). On the other hand, we had an opportunity to measure WB 5-HT levels in depressive subjects (3 males and 4 females, age 29–46 years) with a treatment of selective serotonin reuptake inhibitors (SSRIs; Paxil, Luvox or Depromel). These subjects showed an extremely lower WB 5-HT level (14.1 ± 3.8 ng/ml), as compared to healthy subjects. This may be explained by the mechanism that SSRIs could inhibit 5-HT transporters located at blood-brain barrier as well as pre-synaptic terminals of 5-HT neurons. doi:10.1016/j.neures.2010.07.2094
P1-b20 Conditional Phgdh deletion results in reduced D,Lserine levels and alters monoamine metabolism in the postnatal brain Shozo Tomonaga 1 , Kayoko Esaki 2 , Akira Wada 2 , Satoko Yang 2 , Toshihisa Ohshima 3 , Mitsuhiro Sayano 2 , JungHoon Furuse 4 , Yoshio Hirabayashi 5 , Shigeki Furuya 2 1
Facul Agr, Kyushu Univ, Fukuoka 2 Facul Agr, Kyushu Univ, Fukuoka 3 Facul Agr, Kyushu Univ, Fukuoka 4 Facul Agr, Kyushu Univ, Fukuoka 5 RIKEN BSI, Wako The mammalian brain contains D-serine at high levels. The amino acid is synthesized from L-serine by serine racemase, and it functions as an obligatory coagonist of NMDA receptors. We previously revealed that conditional deletion of brain Phgdh, which catalyzes the first step of L-serine synthesis, resulted in reduced D, L-serine levels in the adult brain. In the present study, we found significant decreases in D- and L-serine contents in the postnatal brain of this conditional knockout mouse. Furthermore, metabolism of monoamines is altered simultaneously in particular regions in the postnatal brain. These results raise the possibility that the availability of L-serine may play a regulatory role in an interaction between glutamatergic and monoamine systems probably via D-serine during the postnatal developmental period. doi:10.1016/j.neures.2010.07.2095
e119
P1-b22 Phosphorylation of Cyclin-dependent kinase 5 at Tyrosine 15 Hiroyuki Kobayashi , Ko Sato, Tomohisa Hosokawa, Taro Saitou, Shin-Ichi Hisanaga Molecular Neurobiology, Department of Biological Sciences Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase activated by binding to brain specific regulatory subunit p35 or p39. Different from other active Cdks in proliferating cells (Cycling Cdks), which require the phosphorylation at Thr161 in the activation loop for activation, Cdk5 is activated only by binding to p35 or p39. Furthermore, in contrast to cycling Cdks, whose kinase activity is inhibited by phosphorylation at tyrosine 15 (Tyr15), Cdk5 is activated by phosphorylation at Tyr15 with Src family kinases or receptor type tyrosine kinases. Activation of Cdk5-p35 by Tyr15 phosphorylation is indicated to induce neurite retraction and spine formation. Thus, phosphorylation of Cdk5 at Tyr15 appears to play an important role in several neuronal activities. However, it is not known well how Tyr15 phosphorylation activates Cdk5. In this study, we have investigated phosphorylation of Cdk5 at Tyr15 using Src family kinases. We transfected Cdk5 and p35 together with Fyn into COS-7 cells and observed phosphorylation of Cdk5 using anti-phospho-Tyr15. However, we could not detect the reaction even though constitutive active Fyn was coexpressed. Unexpectedly, phosphorylation of Cdk5 at Tyr15 was found when Cdk5 alone was expressed in COS-7 cells with active Fyn. We suspected that active Cdk5-p35 inhibits Fyn kinase, and transfected a kinase negative Cdk5 mutant (D144N) with Fyn. Tyr15 phosphorylation was still diminished by coexpression with p35. We wondered the N-terminal region of p35 impairs Tyr15 phosphorylation sterically and coexpressed with p25, N-terminal truncation form of p25. But phosphorylation of Cdk5 was also inhibited by p25. These results suggest that Cdk5 is phosphorylated at Tyr15 only when it is a monomeric free form. These results claim the re-evaluation of Tyr15 phosphorylation on Cdk5 activation. doi:10.1016/j.neures.2010.07.2097
P1-b23 Transactivation of ErbB receptor by GnRH via CaM kinase II in cultured hypothalamic neurons Hideyuki Yamamoto 1 , Sayomi Higa-Nakamine 1 , Noriko Maeda 1 , Tomoko Yamamoto 1 , Seikichi Toku 1 , Masahiro Kawahara 2 1
P1-b21 Effect of bisphenol A exposure during gestation and lactation on amino acid metabolism in the rat brain of offspring Hirokazu Mizokawa Tomoyuki Kanamatsu
, George
Kokubu, Shinichi
Sakamoto,
Department of Environmental Engineering for Symbiosis, Soka university, Tokyo, Japan Exposure to bisphenol A (BPA) during the gestation and lactation periods alters the locomotor activity and learning ability of mature rat offspring. The influences of BPA are caused through estrogen receptor. Estrogen is involved in organogenesis period of brain. The purpose of this study was to elucidate the effect of BPA on amino acid metabolism in the rat brain. The BPA-exposure groups were treated with 0.1, 1.0, and 5.0 ppm BPA in drinking water, respectively, and control group was given tap water. All weaning rats were kept giving the tap water. In this study, male offspring rats between 18 and 24 weeks of age were used. The rats were killed with the microwave treatment under anesthesia 20 min after the injection of [1-13 C]glucose via the tail vein. Brain regions were dissected into frontal cortex, thalamus, striatum, hippocampus and brain stem according to the method of Glowinski and Iverson, then, the amino acid fraction was extracted from each tissue. The extracted fractions were analyzed by 13 C-NMR spectroscopy. The 13 C incorporation into GABA C2 from [1-13 C]glucose was significantly lower in the thalamus and higher in the striatum in 0.1 ppm BPA-exposed group compared to the control group. In other brain regions, there was no significant difference in the 13 C incorporation between four groups. In GABA concentration, there was no significant difference in the thalamus, and a significant increase was shown in the striatum. These results suggest that exposure to BPA during the gestation and lactation may affect GABA (inhibitory neurotransmitter) metabolism in the brain of offspring. An alteration of GABA metabolism may cause the behavioral abnormality in the rat offspring. doi:10.1016/j.neures.2010.07.2096
Dept Biochem, University of the Ryukyus, Nishihara, Japan 2 Dept of Analytical Chem, Kyusyu Univ. of Health and Welfare, Miyazaki, Japan Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic neurons (GnRH neurons). It has been reported that GnRH neurons have GnRH receptor and that the autocrine action of GnRH regulates the functions of GnRH neurons. We have reported that treatment of the immortalized GnRH neurons (GT1-7 cells) with GnRH activated MAP kinase via activation of CaM kinase II. In the present study, we found that activation of MAP kinase was inhibited both by AG1478, an inhibitor of ErbB receptor tyrosine kinase family, and by GM6001, an inhibitor of matrix metalloproteinases. In contrast, TAPI-2, an inhibitor of TACE had no effects on MAP kinase activation. Immunoblot analysis and sequence after RT-PCR indicated that ErbB4, but not epidermal growth factor receptor, was expressed in GT1-7 cells. These results suggested that the activation of CaM kinase II by GnRH induced the activation of MAP kinase via transactivation of ErbB4 in GT1-7 cells. doi:10.1016/j.neures.2010.07.2098
P1-b25 G protein Rho and Rho-kinase regulate the augmenting effect of 5HT on the voltage-dependent Ca2+ current
Noriyuki Watanabe 1 , Satoshi Kawasaki 1 , Shingo Kimura 1 , Misato Harata 1 , Reiko Fujita 2 , Kazuhiko Sasaki 1 1
Dept Physiol, Sch Med, Iwate Med Univ 2 Dept Chemistry, Ctr Lib Arts & Sci, Iwate Med Univ We previously reported that small G protein Rho is involved in the facilitation of the inward current responses to serotonin (5-HT) in the ganglion cells of Aplysia under voltage-clamp. To investigate other role of Rho in the receptor-induced responses in the neuron, we studied action of Rho on the 5-HT-induced facilitation of the voltage-dependent Ca2+ current using conventional two-electrode voltage clamp method in combination with intracellular injection of various enzymes and reagents. We measured a depolarization-induced outward current as indicative of voltage-dependent Ca2+ current to avoid the loss of physiological circumstance. Application of 5-