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Modulation by brain natriuretic peptide of rectify outward K+ current on mouse Schwann cells
Abstracts / Neuroscience Research 58S (2007) S1–S244
P3-c10
Localization of phospholipase C1 on detergentresistant membrane microdomains prepared from synaptic plasma membrane of rat brain
Katsutoshi Taguchi 1 , Haruko Kumanogoh 2 , Shun Nakamura 2 , Shohei Maekawa 1 1 Department of Biosystems Science, Graduate School of Science and Technology, Kobe-University, Kobe, Japan; 2 Division of Biochemistry and Cellular Biology, National Institute of Neuroscience, NCNP, Tokyo, Japan Membrane microdomains (MDs) such as a detergent-resistant membrane microdomain fraction (DRM) have been paid much attention, because many signal-transducing molecules are recovered in the fraction. In order to identify neuronal MD-proteins, monoclonal antibodies against the DRM-components prepared from synaptic plasma membrane of rat brain were produced. One of the antigens reacted with two closely positioned bands of about 140 kDa in SDS-PAGE and the antigen showed agedependent localization on DRM. The antigen was immunoprecipitated and identified as phospholipase C1 through mass analysis and Western blotting. Lipid composition of PIP2 -containing liposomes affected the enzymatic activity and addition of NAP-22, a neuronal MD-protein, inhibited the activity. These results suggest the presence of MDs that perform important roles in neuronal functions through the lipid metabolism.
S197
P3-c13 Modulation by brain natriuretic peptide of rectify outward K+ current on mouse Schwann cells Xiao-Wei Fei School of Life Sciences, Fudan University, Shanghai, China Here we found that mouse Schwann cells expressed all the natriuretic peptide receptors (NPRA, NPRB and NPRC) by molecular techniques and immunohistochemistry. Moreover, extracellular application of BNP, one type of natriuretic peptides, could increase rectify outward K+ current (Ik ) on mouse Schwann cell, which was not concentration-dependent (5–200 nM). BNP also had a significant effect on the kinetic parameters of the steady-state activation. Extracellular application of c-ANF, the NPR-C selective agonist, did not mimic the effect of BNP, while anatin, the blocker of NPR-A, could inhibit the effect. All this indicated that only the receptor NPR-A was involved. With the application of HS-142-1 or 8Br-cGMP, we further confirmed that only NPR-A and its intracellular cGMP pathway took part in the current-increased effect. By means of proliferation assay, BNP seemed to have facilitated effect on Schwann cell proliferation. All the results help us to investigate in terms of therapeutic applications of BNP and basic research of Schwann cell. Research funds: Research grants from Hua-Shan Hospital, Fudan University, Shanghai, China
Research funds: KAKENHI (16370062)
P3-c11 DARPP-32-dependent regulation of spinophilin Ser94 phosphorylation in neostriatal neurons of direct and indirect pathways Uematsu 1,2,3 ,
Maeda 2 ,
Nishi 1
Ken Hisao Akinori Department of Pharmacology, Kurume University, School of Medicine, Kurume, Fukuoka, Japan; 2 Department of Neuropsychiatry, Kurume University, School of Medicine, Kurume, Fukuoka, Japan; 3 St. Marys Hospital, Kurume, Fukuoka, Japan
1
Spinophilin is a protein phosphatase-1 (PP-1)- and actin-binding protein that is enriched in dendritic spines. Spinophilin, by binding to actin filaments, anchors PP-1 in the vicinity of AMPA and NMDA receptors, and thereby modulates excitatory synaptic transmission. Binding of spinophilin to actin is regulated by phosphorylation of the actin-binding domain of spinophilin by cAMP-dependent protein kinase (PKA). Activation of PKA by forskolin stimulated the phosphorylation of spinophilin at Ser94. In this study, we investigated the regulation of spinophilin phosphorylation at Ser94 using mouse neostriatal slices. Activation of dopamine D1 and adenosine A2A receptors increased, whereas activation of dopamine D2 and NMDA receptors decreased, spinophilin Ser94 phosphorylation. D1, D2 and A2A receptors, not NMDA receptors, required DARPP-32 to elicit their effects on spinophilin Ser94 phosphorylation.
P3-c14 Lysophosphatidic acid stimulates astrocyte proliferation through LPA1 Yuki Sogo 1 , Shano Shinya 1 , Ryutaro Moriyama 1 , Jerold Chun 2 , Nobuyuki Fukushima 1 1 Department of Life Science, Kinki University, Japan; 2 The Scripps Research Institute, Ja Lolla, CA, USA Lysophosphatidic acid (LPA) is a signalling phospholipid that regulates the nervous system functions through LPA receptors. Here we explored the role of LPA receptor subtypes in cortical astrocyte functions. Astrocytes cultured under serum-free conditions were found to express the genes of five LPA receptor subtypes, lpa1 to lpa5 . When astrocytes were treated with dibutyryl cyclic adenosine monophosphate, a reagent inducing astrocyte differentiation, lpa1 expression levels remained unchanged, but those of other LPA receptor subtypes were relatively reduced. LPA stimulated DNA synthesis in both undifferentiated and differentiated astrocytes, but failed to do so in astrocytes prepared from lpa1 -null mice. By contrast, LPA-induced inhibition of [3 H]-glutamate uptake was observed in not only both undifferentiated and differentiated astrocytes, but also in lpa1 -deficient astrocytes. These indicate that LPA1 mediates LPA-induced stimulation of DNA synthesis but not inhibition of glutamate uptake in astrocytes. Research funds: Kyowa Hakko Kogyo Co. Ltd.
P3-c12 Neurofibrillary tangles in Lafora disease: Functional
P3-c15 Signaling mechanism inducing deleterious factors in
link between laforin and tau proteins
microglia
Rajat Puri 1 , Kazuhiro Yamakawa 2 , Ganesh Subramaniam 1 1 Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, India; 2 Laboratory for Neurogenetics, RIKEN Brain Science Institute, Wako-shi, Japan
Yukihisa Yosino 1 , Yoko Tohyama 1 , Shinichi Kohsaka 2 , Tadashi Kurihara 1 , Kazuyuki Nakajima 1,2 1 Department of Bioengineering, University of Soka, Tokyo, Japan; 2 National Institute of Neuroscience, Japan
Mutations in EPM2A in human and mice leads to a neurodegenerative condition called as Lafora disease (LD). EPM2A encodes a protein phosphatase known as laforin. Our analyses on the Epm2a gene knockout mice reveal the presence of hyper-phosphorylated tau as neurofibrillary tangles (NFT’s), a hallmark of Alzheimers disease. One of the kinases that phosphorylate tau is GSK3. Intriguingly, the phosphorylation levels of Ser-9 residue of GSK3and the Ser-473 of AKT are lower in the Epm2a knockout mice. AKT is a serine–threonine kinase that phosphorylates and regulates the activity of GSK3. Dephosphorylation of residues Ser-9 of GSK3 and Ser-473 of AKT is known to activate and inactivate these two enzymes, respectively. In this presentation we would discuss the molecular links between laforin and tau proteins and propose that NFT’s might underlie some of the symptoms associated with LD.
To clarify the molecular mechanism by which harmful molecules are induced in infected microglia, we investigated the effects of mitogenactivated protein kinase (MAPK) inhibitors and antioxidant reagent on the production of superoxide anion (SA), nitric oxide (NO) and tumor necrosis factor alpha (TNF␣) in lipopolysaccharide (LPS)-activated microglia. The inhibitor experiments indicated that two MAPKs, c-Jun N-terminal kinase (JNK) and p38MAPK are strongly associated with the induction of NO and TNF␣ in LPS-activated microglia. On the other hand, antioxidant N-acetyl cystein (NAC) was found to suppress the production of not only SA but also NO and TNF␣ in LPS-activated microglia. The antioxidant reagent also significantly reduced LPS-dependent activation of JNK and p38MAPK. Therefore, these results suggested that SA produced in infected microglia induces NO and TNF␣ by activating JNK and p38MAPK.
Research funds: LSRB
P3-c10
Localization of phospholipase C1 on detergentresistant membrane microdomains prepared from synaptic plasma membrane of rat brain
Katsutoshi Taguchi 1 , Haruko Kumanogoh 2 , Shun Nakamura 2 , Shohei Maekawa 1 1 Department of Biosystems Science, Graduate School of Science and Technology, Kobe-University, Kobe, Japan; 2 Division of Biochemistry and Cellular Biology, National Institute of Neuroscience, NCNP, Tokyo, Japan Membrane microdomains (MDs) such as a detergent-resistant membrane microdomain fraction (DRM) have been paid much attention, because many signal-transducing molecules are recovered in the fraction. In order to identify neuronal MD-proteins, monoclonal antibodies against the DRM-components prepared from synaptic plasma membrane of rat brain were produced. One of the antigens reacted with two closely positioned bands of about 140 kDa in SDS-PAGE and the antigen showed agedependent localization on DRM. The antigen was immunoprecipitated and identified as phospholipase C1 through mass analysis and Western blotting. Lipid composition of PIP2 -containing liposomes affected the enzymatic activity and addition of NAP-22, a neuronal MD-protein, inhibited the activity. These results suggest the presence of MDs that perform important roles in neuronal functions through the lipid metabolism.
S197
P3-c13 Modulation by brain natriuretic peptide of rectify outward K+ current on mouse Schwann cells Xiao-Wei Fei School of Life Sciences, Fudan University, Shanghai, China Here we found that mouse Schwann cells expressed all the natriuretic peptide receptors (NPRA, NPRB and NPRC) by molecular techniques and immunohistochemistry. Moreover, extracellular application of BNP, one type of natriuretic peptides, could increase rectify outward K+ current (Ik ) on mouse Schwann cell, which was not concentration-dependent (5–200 nM). BNP also had a significant effect on the kinetic parameters of the steady-state activation. Extracellular application of c-ANF, the NPR-C selective agonist, did not mimic the effect of BNP, while anatin, the blocker of NPR-A, could inhibit the effect. All this indicated that only the receptor NPR-A was involved. With the application of HS-142-1 or 8Br-cGMP, we further confirmed that only NPR-A and its intracellular cGMP pathway took part in the current-increased effect. By means of proliferation assay, BNP seemed to have facilitated effect on Schwann cell proliferation. All the results help us to investigate in terms of therapeutic applications of BNP and basic research of Schwann cell. Research funds: Research grants from Hua-Shan Hospital, Fudan University, Shanghai, China
Research funds: KAKENHI (16370062)
P3-c11 DARPP-32-dependent regulation of spinophilin Ser94 phosphorylation in neostriatal neurons of direct and indirect pathways Uematsu 1,2,3 ,
Maeda 2 ,
Nishi 1
Ken Hisao Akinori Department of Pharmacology, Kurume University, School of Medicine, Kurume, Fukuoka, Japan; 2 Department of Neuropsychiatry, Kurume University, School of Medicine, Kurume, Fukuoka, Japan; 3 St. Marys Hospital, Kurume, Fukuoka, Japan
1
Spinophilin is a protein phosphatase-1 (PP-1)- and actin-binding protein that is enriched in dendritic spines. Spinophilin, by binding to actin filaments, anchors PP-1 in the vicinity of AMPA and NMDA receptors, and thereby modulates excitatory synaptic transmission. Binding of spinophilin to actin is regulated by phosphorylation of the actin-binding domain of spinophilin by cAMP-dependent protein kinase (PKA). Activation of PKA by forskolin stimulated the phosphorylation of spinophilin at Ser94. In this study, we investigated the regulation of spinophilin phosphorylation at Ser94 using mouse neostriatal slices. Activation of dopamine D1 and adenosine A2A receptors increased, whereas activation of dopamine D2 and NMDA receptors decreased, spinophilin Ser94 phosphorylation. D1, D2 and A2A receptors, not NMDA receptors, required DARPP-32 to elicit their effects on spinophilin Ser94 phosphorylation.
P3-c14 Lysophosphatidic acid stimulates astrocyte proliferation through LPA1 Yuki Sogo 1 , Shano Shinya 1 , Ryutaro Moriyama 1 , Jerold Chun 2 , Nobuyuki Fukushima 1 1 Department of Life Science, Kinki University, Japan; 2 The Scripps Research Institute, Ja Lolla, CA, USA Lysophosphatidic acid (LPA) is a signalling phospholipid that regulates the nervous system functions through LPA receptors. Here we explored the role of LPA receptor subtypes in cortical astrocyte functions. Astrocytes cultured under serum-free conditions were found to express the genes of five LPA receptor subtypes, lpa1 to lpa5 . When astrocytes were treated with dibutyryl cyclic adenosine monophosphate, a reagent inducing astrocyte differentiation, lpa1 expression levels remained unchanged, but those of other LPA receptor subtypes were relatively reduced. LPA stimulated DNA synthesis in both undifferentiated and differentiated astrocytes, but failed to do so in astrocytes prepared from lpa1 -null mice. By contrast, LPA-induced inhibition of [3 H]-glutamate uptake was observed in not only both undifferentiated and differentiated astrocytes, but also in lpa1 -deficient astrocytes. These indicate that LPA1 mediates LPA-induced stimulation of DNA synthesis but not inhibition of glutamate uptake in astrocytes. Research funds: Kyowa Hakko Kogyo Co. Ltd.
P3-c12 Neurofibrillary tangles in Lafora disease: Functional
P3-c15 Signaling mechanism inducing deleterious factors in
link between laforin and tau proteins
microglia
Rajat Puri 1 , Kazuhiro Yamakawa 2 , Ganesh Subramaniam 1 1 Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, India; 2 Laboratory for Neurogenetics, RIKEN Brain Science Institute, Wako-shi, Japan
Yukihisa Yosino 1 , Yoko Tohyama 1 , Shinichi Kohsaka 2 , Tadashi Kurihara 1 , Kazuyuki Nakajima 1,2 1 Department of Bioengineering, University of Soka, Tokyo, Japan; 2 National Institute of Neuroscience, Japan
Mutations in EPM2A in human and mice leads to a neurodegenerative condition called as Lafora disease (LD). EPM2A encodes a protein phosphatase known as laforin. Our analyses on the Epm2a gene knockout mice reveal the presence of hyper-phosphorylated tau as neurofibrillary tangles (NFT’s), a hallmark of Alzheimers disease. One of the kinases that phosphorylate tau is GSK3. Intriguingly, the phosphorylation levels of Ser-9 residue of GSK3and the Ser-473 of AKT are lower in the Epm2a knockout mice. AKT is a serine–threonine kinase that phosphorylates and regulates the activity of GSK3. Dephosphorylation of residues Ser-9 of GSK3 and Ser-473 of AKT is known to activate and inactivate these two enzymes, respectively. In this presentation we would discuss the molecular links between laforin and tau proteins and propose that NFT’s might underlie some of the symptoms associated with LD.
To clarify the molecular mechanism by which harmful molecules are induced in infected microglia, we investigated the effects of mitogenactivated protein kinase (MAPK) inhibitors and antioxidant reagent on the production of superoxide anion (SA), nitric oxide (NO) and tumor necrosis factor alpha (TNF␣) in lipopolysaccharide (LPS)-activated microglia. The inhibitor experiments indicated that two MAPKs, c-Jun N-terminal kinase (JNK) and p38MAPK are strongly associated with the induction of NO and TNF␣ in LPS-activated microglia. On the other hand, antioxidant N-acetyl cystein (NAC) was found to suppress the production of not only SA but also NO and TNF␣ in LPS-activated microglia. The antioxidant reagent also significantly reduced LPS-dependent activation of JNK and p38MAPK. Therefore, these results suggested that SA produced in infected microglia induces NO and TNF␣ by activating JNK and p38MAPK.
Research funds: LSRB