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1.
Neurochem Res ; 47(9): 2632-2644, 2022 Sep.
Article in English | MEDLINE | ID: mdl-34984589

ABSTRACT

Neuronal differentiation, maturation, and synapse formation are regulated by various growth factors. Here we show that epidermal growth factor (EGF) negatively regulates presynaptic maturation and synapse formation. In cortical neurons, EGF maintained axon elongation and reduced the sizes of growth cones in culture. Furthermore, EGF decreased the levels of presynaptic molecules and number of presynaptic puncta, suggesting that EGF inhibits neuronal maturation. The reduction of synaptic sites is confirmed by the decreased frequencies of miniature EPSCs. In vivo analysis revealed that while peripherally administrated EGF decreased the levels of presynaptic molecules and numbers of synaptophysin-positive puncta in the prefrontal cortices of neonatal rats, EGF receptor inhibitors upregulated these indexes, suggesting that endogenous EGF receptor ligands suppress presynaptic maturation. Electron microscopy further revealed that EGF decreased the numbers, but not the sizes, of synaptic structures in vivo. These findings suggest that endogenous EGF and/or other EGF receptor ligands negatively modulates presynaptic maturation and synapse formation.


Subject(s)
Epidermal Growth Factor , Synapses , Animals , Axons , Cells, Cultured , Epidermal Growth Factor/pharmacology , Neurogenesis/physiology , Neurons/metabolism , Rats , Synapses/metabolism
2.
Neuropsychopharmacology ; 41(3): 802-10, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26171716

ABSTRACT

Calsyntenin-2 has an evolutionarily conserved role in cognition. In a human genome-wide screen, the CLSTN2 locus was associated with verbal episodic memory, and expression of human calsyntenin-2 rescues the associative learning defect in orthologous Caenorhabditis elegans mutants. Other calsyntenins promote synapse development, calsyntenin-1 selectively of excitatory synapses and calsyntenin-3 of excitatory and inhibitory synapses. We found that targeted deletion of calsyntenin-2 in mice results in a selective reduction in functional inhibitory synapses. Reduced inhibitory transmission was associated with a selective reduction of parvalbumin interneurons in hippocampus and cortex. Clstn2(-/-) mice showed normal behavior in elevated plus maze, forced swim test, and novel object recognition assays. However, Clstn2(-/-) mice were hyperactive in the open field and showed deficits in spatial learning and memory in the Morris water maze and Barnes maze. These results confirm a function for calsyntenin-2 in cognitive performance and indicate an underlying mechanism that involves parvalbumin interneurons and aberrant inhibitory transmission.


Subject(s)
Calcium-Binding Proteins/deficiency , Cognition Disorders/metabolism , Interneurons/metabolism , Membrane Proteins/deficiency , Synaptic Transmission/physiology , gamma-Aminobutyric Acid/metabolism , Animals , Brain/metabolism , Calcium-Binding Proteins/genetics , Exploratory Behavior/physiology , Female , Fluorescent Antibody Technique , Male , Maze Learning/physiology , Membrane Proteins/genetics , Mice, Inbred C57BL , Mice, Knockout , Motor Activity/physiology , Neural Inhibition/physiology , Parvalbumins/metabolism , Recognition, Psychology/physiology , Spatial Memory/physiology , Tissue Culture Techniques
3.
Neuron ; 80(1): 113-28, 2013 Oct 02.
Article in English | MEDLINE | ID: mdl-24094106

ABSTRACT

Perturbations of cell surface synapse-organizing proteins, particularly α-neurexins, contribute to neurodevelopmental and psychiatric disorders. From an unbiased screen, we identify calsyntenin-3 (alcadein-ß) as a synapse-organizing protein unique in binding and recruiting α-neurexins, but not ß-neurexins. Calsyntenin-3 is present in many pyramidal neurons throughout cortex and hippocampus but is most highly expressed in interneurons. The transmembrane form of calsyntenin-3 can trigger excitatory and inhibitory presynapse differentiation in contacting axons. However, calsyntenin-3-shed ectodomain, which represents about half the calsyntenin-3 pool in brain, suppresses the ability of multiple α-neurexin partners including neuroligin 2 and LRRTM2 to induce presynapse differentiation. Clstn3⁻/⁻ mice show reductions in excitatory and inhibitory synapse density by confocal and electron microscopy and corresponding deficits in synaptic transmission. These results identify calsyntenin-3 as an α-neurexin-specific binding partner required for normal functional GABAergic and glutamatergic synapse development.


Subject(s)
Calcium-Binding Proteins/metabolism , Hippocampus/cytology , Membrane Proteins/metabolism , Neurons/metabolism , Synapses/metabolism , Synaptic Transmission/physiology , Animals , Calcium-Binding Proteins/genetics , Cell Adhesion Molecules, Neuronal/metabolism , Cell Differentiation/physiology , Cells, Cultured , Cerebral Cortex/growth & development , Cerebral Cortex/pathology , Hippocampus/growth & development , Hippocampus/metabolism , Hippocampus/ultrastructure , Humans , Membrane Proteins/genetics , Nerve Tissue Proteins/metabolism , Neural Cell Adhesion Molecules/metabolism , Neurons/cytology , Rats , Receptors, Cell Surface/metabolism , Synapses/genetics
4.
J Cell Biol ; 200(3): 321-36, 2013 Feb 04.
Article in English | MEDLINE | ID: mdl-23358245

ABSTRACT

Rare variants in MDGAs (MAM domain-containing glycosylphosphatidylinositol anchors), including multiple protein-truncating deletions, are linked to autism and schizophrenia, but the function of these genes is poorly understood. Here, we show that MDGA1 and MDGA2 bound to neuroligin-2 inhibitory synapse-organizing protein, also implicated in neurodevelopmental disorders. MDGA1 inhibited the synapse-promoting activity of neuroligin-2, without altering neuroligin-2 surface trafficking, by inhibiting interaction of neuroligin-2 with neurexin. MDGA binding and suppression of synaptogenic activity was selective for neuroligin-2 and not neuroligin-1 excitatory synapse organizer. Overexpression of MDGA1 in cultured rat hippocampal neurons reduced inhibitory synapse density without altering excitatory synapse density. Furthermore, RNAi-mediated knockdown of MDGA1 selectively increased inhibitory but not excitatory synapse density. These results identify MDGA1 as one of few identified negative regulators of synapse development with a unique selectivity for inhibitory synapses. These results also place MDGAs in the neurexin-neuroligin synaptic pathway implicated in neurodevelopmental disorders and support the idea that an imbalance between inhibitory and excitatory synapses may contribute to these disorders.


Subject(s)
Autistic Disorder/metabolism , Cell Adhesion Molecules, Neuronal/metabolism , Nerve Tissue Proteins/metabolism , Neural Cell Adhesion Molecules/metabolism , Neural Inhibition , Synapses/metabolism , Animals , COS Cells , Chlorocebus aethiops , Dendrites/metabolism , GPI-Linked Proteins , Gene Knockdown Techniques , HEK293 Cells , Humans , Mice , Models, Biological , Neural Cell Adhesion Molecules/chemistry , Protein Binding , Protein Structure, Tertiary , Rats , Recombinant Proteins
5.
J Neurochem ; 118(1): 45-56, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21517852

ABSTRACT

Although epidermal growth factor (EGF) receptor (ErbB1) is implicated in Parkinson's disease and schizophrenia, the neurotrophic action of ErbB1 ligands on nigral dopaminergic neurons remains controversial. Here, we ascertained colocalization of ErbB1 and tyrosine hydroxylase (TH) immunoreactivity and then characterized the neurotrophic effects of ErbB1 ligands on this cell population. In mesencephalic culture, EGF and glial-derived neurotrophic factor (GDNF) similarly promoted survival and neurite elongation of dopaminergic neurons and dopamine uptake. The EGF-promoted dopamine uptake was not inhibited by GDNF-neutralizing antibody or TrkB-Fc, whereas EGF-neutralizing antibody fully blocked the neurotrophic activity of the conditioned medium that was prepared from EGF-stimulated mesencephalic cultures. The neurotrophic action of EGF was abolished by ErbB1 inhibitors and genetic disruption of erbB1 in culture. In vivo administration of ErbB1 inhibitors to rat neonates diminished TH and dopamine transporter (DAT) levels in the striatum and globus pallidus but not in the frontal cortex. In parallel, there was a reduction in the density of dopaminergic varicosities exhibiting intense TH immunoreactivity. In agreement, postnatal erbB1-deficient mice exhibited similar decreases in TH levels. Although neurotrophic supports to dopaminergic neurons are redundant, these results confirm that ErbB1 ligands contribute to the phenotypic and functional development of nigral dopaminergic neurons.


Subject(s)
Dopamine/metabolism , ErbB Receptors/metabolism , Mesencephalon , Neurons/physiology , Signal Transduction/physiology , Animals , Animals, Newborn , Cells, Cultured , Drug Interactions , Embryo, Mammalian , Enzyme Inhibitors/pharmacology , Epidermal Growth Factor/pharmacology , ErbB Receptors/deficiency , Female , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/genetics , Glial Cell Line-Derived Neurotrophic Factor/pharmacology , In Vitro Techniques , Male , Mesencephalon/cytology , Mesencephalon/embryology , Mesencephalon/growth & development , Mice , Mice, Knockout , Neurites/drug effects , Neurites/physiology , Neurons/cytology , Neurons/drug effects , Pregnancy , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Signal Transduction/genetics , Time Factors , Tyrosine 3-Monooxygenase/metabolism
6.
Hum Genet ; 130(4): 563-73, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21424692

ABSTRACT

Growing genetic evidence is converging in favor of common pathogenic mechanisms for autism spectrum disorders (ASD), intellectual disability (ID or mental retardation) and schizophrenia (SCZ), three neurodevelopmental disorders affecting cognition and behavior. Copy number variations and deleterious mutations in synaptic organizing proteins including NRXN1 have been associated with these neurodevelopmental disorders, but no such associations have been reported for NRXN2 or NRXN3. From resequencing the three neurexin genes in individuals affected by ASD (n = 142), SCZ (n = 143) or non-syndromic ID (n = 94), we identified a truncating mutation in NRXN2 in a patient with ASD inherited from a father with severe language delay and family history of SCZ. We also identified a de novo truncating mutation in NRXN1 in a patient with SCZ, and other potential pathogenic ASD mutations. These truncating mutations result in proteins that fail to promote synaptic differentiation in neuron coculture and fail to bind either of the established postsynaptic binding partners LRRTM2 or NLGN2 in cell binding assays. Our findings link NRXN2 disruption to the pathogenesis of ASD for the first time and further strengthen the involvement of NRXN1 in SCZ, supporting the notion of a common genetic mechanism in these disorders.


Subject(s)
Cell Adhesion Molecules, Neuronal/genetics , Child Development Disorders, Pervasive/genetics , Mutation/genetics , Nerve Tissue Proteins/genetics , Schizophrenia/genetics , Amino Acid Sequence , Animals , COS Cells , Calcium-Binding Proteins , Case-Control Studies , Cell Adhesion Molecules, Neuronal/metabolism , Cells, Cultured , Child , Chlorocebus aethiops , Cohort Studies , Female , Gene Dosage , Genetic Predisposition to Disease , Humans , Language Development Disorders/genetics , Male , Membrane Proteins/metabolism , Mice , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , Neural Cell Adhesion Molecules , Neurons/cytology , Neurons/metabolism , Pedigree , Sequence Homology, Amino Acid
7.
World J Biol Chem ; 1(5): 133-43, 2010 May 26.
Article in English | MEDLINE | ID: mdl-21540998

ABSTRACT

Brain-derived neurotrophic factor (BDNF), a critical neurotrophin, regulates many neuronal aspects including cell differentiation, cell survival, neurotransmission, and synaptic plasticity in the central nervous system (CNS). Though BDNF has two types of receptors, high affinity tropomyosin-related kinase (Trk)B and low affinity p75 receptors, BDNF positively exerts its biological effects on neurons via activation of TrkB and of resultant intracellular signaling cascades including mitogen-activated protein kinase/extracellular signal-regulated protein kinase, phospholipase Cγ, and phosphoinositide 3-kinase pathways. Notably, it is possible that alteration in the expression and/or function of BDNF in the CNS is involved in the pathophysiology of various brain diseases such as stroke, Parkinson's disease, Alzheimer's disease, and mental disorders. On the other hand, glucocorticoids, stress-induced steroid hormones, also putatively contribute to the pathophysiology of depression. Interestingly, in addition to the reduction in BDNF levels due to increased glucocorticoid exposure, current reports demonstrate possible interactions between glucocorticoids and BDNF-mediated neuronal functions. Other steroid hormones, such as estrogen, are involved in not only sexual differentiation in the brain, but also numerous neuronal events including cell survival and synaptic plasticity. Furthermore, it is well known that estrogen plays a role in the pathophysiology of Parkinson's disease, Alzheimer's disease, and mental illness, while serving to regulate BDNF expression and/or function. Here, we present a broad overview of the current knowledge concerning the association between BDNF expression/function and steroid hormones (glucocorticoids and estrogen).

8.
Endocrinology ; 148(2): 627-37, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17082253

ABSTRACT

Although many studies have suggested that estrogen acts as a neuroprotective agent in oxidative stress, the underlying mechanism has not been fully elucidated. In the present study, we examined the effect of 17beta-estradiol (17beta-E2) on H(2)O(2)-induced death signaling in cultured cortical neurons. Exposure of the cortical neurons to H(2)O(2) triggered a series of events, including overactivation of p44/42 MAPK and intracellular Ca(2+) accumulation via voltage-gated Ca(2+) channels and ionotropic glutamate receptors, resulting in apoptotic-like cell death. The MAPK pathway might work as death signaling in our system, because the MAPK pathway inhibitor, U0126, blocked H(2)O(2)-induced MAPK activation, Ca(2+) overload, and cell death. Interestingly, a similar inhibitory effect on H(2)O(2)-triggered MAPK activation, Ca(2+) accumulation, and cell death was observed in cultures incubated with 17beta-E2 for 24 h before exposure to H(2)O(2), suggesting that the protective effect of 17beta-E2 is induced via attenuating overactivation of the MAPK pathway. Furthermore, we found that ionotropic glutamate receptor subunits, including NR2A and GluR2/3, but not NR2B and GluR1, were down-regulated in the 17beta-E2-treated cultures. The down-regulation of these glutamate receptor subunits was also observed after chronic treatment with U0126. Therefore, it is possible that 17beta-E2 down-regulates the expression of the ionotropic glutamate receptors by reducing activity of the MAPK pathway, which might be important for the protective effect of 17beta-E2 against oxidative stress-induced toxicity.


Subject(s)
Calcium/metabolism , Cerebral Cortex/physiology , Estradiol/pharmacology , Intracellular Membranes/metabolism , Mitogen-Activated Protein Kinases/metabolism , Neuroprotective Agents/pharmacology , Oxidative Stress/physiology , Animals , Butadienes/pharmacology , Calcium Channels/metabolism , Cell Death/drug effects , Cell Death/physiology , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/metabolism , Down-Regulation , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Hydrogen Peroxide/pharmacology , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Neurons/drug effects , Neurons/physiology , Nitriles/pharmacology , Oxidants/pharmacology , Rats , Receptors, Estrogen/physiology , Receptors, Glutamate/metabolism
9.
Mol Cell Neurosci ; 31(1): 70-84, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16214365

ABSTRACT

The mechanisms underlying BDNF-modulated neurotransmitter release remain elusive. Here, we found that 24-h exposure of postnatal cortical neurons to BDNF potentiated depolarization-evoked glutamate and GABA release in a protein synthesis-dependent manner. BDNF-potentiated glutamate release occurred through the PLC-gamma and MAPK pathways. The expression of synapsin I, synaptotagmin, and synaptophysin, but not of syntaxin or SNAP25, increased through the PLC-gamma and MAPK pathways. In contrast, BDNF-up-regulated GABA release and GAD65/67 expression depended on MAPK. Furthermore, neuronal activity was necessary for the up-regulation of glutamate release and synapsin I, synaptotagmin, and synaptophysin expression, but not of GABA or GAD65/67. PLC-gamma inhibitor attenuated BDNF-stimulated long-lasting MAPK activation. As BDNF rapidly potentiates glutamatergic transmission through PLC-gamma (J. Biol. Chem. 277, (2002) 6520-6529), PLC-gamma-mediated neuronal activity might sustain MAPK activation, resulting in BDNF-potentiated glutamate release. In conclusion, BDNF potentiates the excitatory and inhibitory system separately, which may be important for the regulation of synaptic plasticity.


Subject(s)
Brain-Derived Neurotrophic Factor/pharmacology , Cerebral Cortex/physiology , Glutamic Acid/metabolism , Neurons/physiology , gamma-Aminobutyric Acid/metabolism , Animals , Animals, Newborn , Cells, Cultured , Cerebral Cortex/drug effects , Immunohistochemistry , Neurons/drug effects , Rats , Rats, Wistar , Signal Transduction/drug effects , Signal Transduction/physiology
10.
Brain Res ; 1038(2): 223-30, 2005 Mar 21.
Article in English | MEDLINE | ID: mdl-15757638

ABSTRACT

We have recently reported that the ASK1-p38 MAPK pathway has an important role in the low potassium (LK)-induced apoptosis of cultured cerebellar granule neurons. In the present study, we observed that ERK1/2 were significantly activated 6 h after a change of medium from HK (high potassium) to LK. In addition, U0126, a specific inhibitor of MEKs, remarkably prevented the apoptosis of cultured cerebellar granule neurons. Then, we examined the mechanism underlying the activation of ERK1/2 in the LK-induced apoptotic pathway. The addition of SB203580, an inhibitor of p38 MAPK, suppressed the increase in the phosphorylation of ERK1/2 after the change to LK medium. Furthermore, we found that the expression of a constitutively active mutant of ASK1, an upstream kinase of p38 MAPK, enhanced the phosphorylation of ERK1/2. These results suggest that ERK1/2 play a crucial role in LK-induced apoptosis of cultured cerebellar granule neurons and that the LK-stimulated activation of ERK1/2 is regulated by the ASK1-p38 MAPK pathway.


Subject(s)
Apoptosis/physiology , Cerebellum/cytology , Extracellular Signal-Regulated MAP Kinases/physiology , MAP Kinase Kinase Kinase 5/physiology , Neurons/physiology , Potassium Deficiency/metabolism , Signal Transduction/physiology , p38 Mitogen-Activated Protein Kinases/physiology , Adenoviridae/genetics , Animals , Blotting, Western , Butadienes/pharmacology , Cells, Cultured , Cerebellum/drug effects , Enzyme Inhibitors/pharmacology , Female , Immunohistochemistry , Male , Mitogen-Activated Protein Kinase 7/genetics , Mitogen-Activated Protein Kinase 7/physiology , Nitriles/pharmacology , Rats , Rats, Wistar , Tetrazolium Salts , Thiazoles
11.
J Biol Chem ; 279(41): 43245-53, 2004 Oct 08.
Article in English | MEDLINE | ID: mdl-15297459

ABSTRACT

Little is known about the role of the integrin-associated protein (IAP, or CD47) in neuronal development and its function in the central nervous system. We investigated neuronal responses in IAP-overexpressing cortical neurons using a virus-gene transfer system. We found that dendritic outgrowth was significantly enhanced in IAP (form 4)-transfected neurons. Furthermore, synaptic proteins including synaptotagmin, syntaxin, synapsin I, and SNAP25 (25-kDa synaptosomal associated protein) were up-regulated. In accordance with this finding, the release of the excitatory transmitter glutamate and the frequencies of Ca2+ oscillations (glutamate-mediated synaptic transmission) were increased. Interestingly, the overexpression of IAP activated mitogen-activated protein kinase (MAPK), and this activation was required for the IAP-dependent biological effects. After down-regulation of the endogenous IAP by small interfering RNA, MAPK activity, synaptic protein levels, and glutamate release decreased. These observations suggest that the IAP plays important roles in dendritic outgrowth and synaptic transmission in developing cortical neurons through the activation of MAPK.


Subject(s)
Antigens, CD/physiology , Cerebral Cortex/metabolism , Neurons/metabolism , Adenoviridae/genetics , Amino Acids/chemistry , Animals , Antigens, CD/chemistry , CD47 Antigen , Calcium/chemistry , Calcium/metabolism , Calcium-Binding Proteins/chemistry , Cells, Cultured , DNA, Complementary/metabolism , Dendrites/metabolism , Down-Regulation , Gene Transfer Techniques , Glutamic Acid/chemistry , Immunohistochemistry , MAP Kinase Signaling System , Membrane Glycoproteins/chemistry , Membrane Proteins/chemistry , Nerve Tissue Proteins/chemistry , Oscillometry , Qa-SNARE Proteins , RNA, Small Interfering/metabolism , Rats , Synaptic Transmission , Synaptosomal-Associated Protein 25 , Synaptosomes/metabolism , Synaptotagmins , Up-Regulation , Viruses/genetics
12.
Brain Res Mol Brain Res ; 119(2): 184-91, 2003 Nov 26.
Article in English | MEDLINE | ID: mdl-14625085

ABSTRACT

On cell maturation following culture in medium containing 26 mM potassium (high K+; HK), a change to medium containing 5 mM potassium (low K+; LK) rapidly induces apoptosis in rat cerebellar granule neurons. Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) have survival-promoting effects on the neurons via PI3-K. However, it remains unclear how they prevent the apoptosis in the pathway downstream of phosphatidylinositol-3 kinase (PI3-K). Recently, we have reported that PI3-K-ASK1 pathway is involved in signal-transduction to p38 MAPK (p38)-c-Jun pathway. Here we found that IGF-1 had a greater survival-promoting effect than BDNF, and activated PI3-K to a higher level and maintained the level for a longer time. BDNF and IGF-1 suppressed the activation of p38 and c-Jun, but not of c-Jun N-terminal kinase (JNK), caused by lowering the potassium concentration. The inhibitory effects of IGF-1 were much greater than those of BDNF. In addition, LY294002, a specific inhibitor of PI3-K, cancelled the inhibitory effects of BDNF and IGF-1. These results suggest that the greater inhibitory effects of IGF-1 than BDNF, on activation of p38 and c-Jun and apoptosis, are caused by the higher level of PI3-K activation during LK-induced apoptosis of cultured cerebellar granule neurons.


Subject(s)
Apoptosis/physiology , Brain-Derived Neurotrophic Factor/metabolism , Insulin-Like Growth Factor I/metabolism , Mitogen-Activated Protein Kinases/metabolism , Neurons/enzymology , Proto-Oncogene Proteins c-jun/metabolism , Animals , Animals, Newborn , Apoptosis/drug effects , Brain-Derived Neurotrophic Factor/pharmacology , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Central Nervous System/cytology , Central Nervous System/enzymology , Central Nervous System/growth & development , Enzyme Inhibitors/pharmacology , Female , Insulin-Like Growth Factor I/pharmacology , JNK Mitogen-Activated Protein Kinases , Male , Mitogen-Activated Protein Kinases/drug effects , Neurons/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Potassium Deficiency/enzymology , Proto-Oncogene Proteins c-jun/drug effects , Rats , Rats, Wistar , Signal Transduction/drug effects , Signal Transduction/physiology , p38 Mitogen-Activated Protein Kinases
13.
J Biol Chem ; 278(42): 41259-69, 2003 Oct 17.
Article in English | MEDLINE | ID: mdl-12902347

ABSTRACT

Very little is known about the contribution of a low affinity neurotrophin receptor, p75, to neurotransmitter release. Here we show that nerve growth factor (NGF) induced a rapid release of glutamate and an increase of Ca2+ in cerebellar neurons through a p75-dependent pathway. The NGF-induced release occurred even in the presence of the Trk inhibitor K252a. The release caused by NGF but not brain-derived neurotrophic factor was enhanced in neurons overexpressing p75. Further, after transfection of p75-small interfering RNA, which down-regulated the endogenous p75 expression, the NGF-induced release was inhibited, suggesting that the NGF-induced glutamate release was through p75. We found that the NGF-increased Ca2+ was derived from the ryanodine-sensitive Ca2+ receptor and that the NGF-increased Ca2+ was essential for the NGF-induced glutamate release. Furthermore, scyphostatin, a sphingomyelinase inhibitor, blocked the NGF-dependent Ca2+ increase and glutamate release, suggesting that a ceramide produced by sphingomyelinase was required for the NGF-stimulated Ca2+ increase and glutamate release. This action of NGF only occurred in developing neurons whereas the brain-derived neurotrophic factor-mediated Ca2+ increase and glutamate release was observed at the mature neuronal stage. Thus, we demonstrate that NGF-mediated neurotransmitter release via the p75-dependent pathway has an important role in developing neurons.


Subject(s)
Calcium/metabolism , Ceramides/metabolism , Cerebellum/metabolism , Egtazic Acid/analogs & derivatives , Glutamic Acid/metabolism , Nerve Growth Factor/metabolism , Neurons/metabolism , Receptors, Nerve Growth Factor/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Adenoviridae/genetics , Amides/pharmacology , Amino Acids/metabolism , Animals , Calcium/pharmacology , Cells, Cultured , Chelating Agents/pharmacology , Dose-Response Relationship, Drug , Down-Regulation , Egtazic Acid/pharmacology , Enzyme Activation , Immunoblotting , Immunohistochemistry , Magnesium/metabolism , Pyrones/pharmacology , Rats , Receptor, Nerve Growth Factor , Sphingomyelin Phosphodiesterase/metabolism , Time Factors , Transfection
14.
Mol Endocrinol ; 17(5): 831-44, 2003 May.
Article in English | MEDLINE | ID: mdl-12554763

ABSTRACT

Changes in synaptic efficacy are considered necessary for learning and memory. Recently, it has been suggested that estrogen controls synaptic function in the central nervous system. However, it is unclear how estrogen regulates synaptic function in central nervous system neurons. We found that estrogen potentiated presynaptic function in cultured hippocampal neurons. Chronic treatment with estradiol (1 or 10 nm) for 24 h significantly increased a high potassium-induced glutamate release. The estrogen-potentiated glutamate release required the activation of both phosphatidylinositol 3-kinase and MAPK. The high potassium-evoked release with or without estradiol pretreatment was blocked by tetanus neurotoxin, which is an inhibitor of exocytosis. In addition, the reduction in intensity of FM1-43 fluorescence, which labeled presynaptic vesicles, was enhanced by estradiol, suggesting that estradiol potentiated the exocytotic mechanism. Furthermore, protein levels of synaptophysin, syntaxin, and synaptotagmin (synaptic proteins, respectively) were up-regulated by estradiol. We confirmed that the up-regulation of synaptophysin was blocked by the MAPK pathway inhibitor, U0126. These results suggested that estrogen enhanced presynaptic function through the up-regulated exocytotic system. In this study, we propose that estrogen reinforced excitatory synaptic transmission via potentiated-glutamate release from presynaptic sites.


Subject(s)
Calcium-Binding Proteins , Cell Polarity/physiology , Estrogens/pharmacology , Glutamic Acid/metabolism , Mitogen-Activated Protein Kinases/metabolism , Neurons/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Animals , Cell Polarity/drug effects , Cells, Cultured , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Estradiol/pharmacology , Estrogen Receptor Modulators/pharmacology , Exocytosis/drug effects , Hippocampus/cytology , Hippocampus/drug effects , Hippocampus/metabolism , Membrane Glycoproteins/drug effects , Membrane Glycoproteins/metabolism , Membrane Proteins/drug effects , Membrane Proteins/metabolism , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/drug effects , Nerve Tissue Proteins/drug effects , Nerve Tissue Proteins/metabolism , Neurons/drug effects , Phosphatidylinositol 3-Kinases/drug effects , Phosphoinositide-3 Kinase Inhibitors , Progesterone/pharmacology , Qa-SNARE Proteins , Rats , Rats, Wistar , Synaptic Transmission/drug effects , Synaptophysin/drug effects , Synaptophysin/metabolism , Synaptotagmins , gamma-Aminobutyric Acid/metabolism
15.
J Neurochem ; 82(2): 249-57, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12124426

ABSTRACT

The death and survival of neuronal cells are regulated by various signaling pathways during development of the brain and in neuronal diseases. Previously, we demonstrated that the neuronal adhesion molecule brain immunoglobulin-like molecule with tyrosine-based activation motifs/SHP substrate 1 (BIT/SHPS-1) is involved in brain-derived neurotrophic factor (BDNF)-promoted neuronal cell survival. Here, we report the apoptosis-inducing effect of CD47/integrin-associated protein (IAP), the heterophilic binding partner of BIT/SHPS-1, on neuronal cells. We generated a recombinant adenovirus vector expressing a neuronal form of CD47/IAP, and found that the expression of CD47/IAP by infection with CD47/IAP adenovirus induced the death of cultured cerebral cortical neurons. The numbers of TdT-mediated biotin-dUTP nick-end labelling (TUNEL)-positive neurons and of cells displaying apoptotic nuclei increased by expression of CD47/IAP. Neuronal cell death was prevented by the addition of the broad-spectrum caspase inhibitor Z-VAD-fmk. Furthermore, we observed that co-expression of CD47/IAP with BIT/SHPS-1 enhanced neuronal cell death, and that BDNF prevented it. These results suggest that CD47/IAP is involved in a novel pathway which regulates caspase-dependent apoptosis of cultured cerebral cortical neurons. CD47/IAP-induced death of cultured cortical neurons may be regulated by the interaction of CD47/IAP with BIT/SHPS-1 and by BDNF.


Subject(s)
Antigens, CD/biosynthesis , Antigens, Differentiation , Apoptosis/physiology , Carrier Proteins/biosynthesis , Cerebral Cortex/metabolism , Neural Cell Adhesion Molecule L1 , Neurons/metabolism , Receptors, Immunologic , Adenoviridae/genetics , Animals , Antigens, CD/genetics , Antigens, CD/pharmacology , Apoptosis/drug effects , CD47 Antigen , Carrier Proteins/genetics , Carrier Proteins/pharmacology , Cell Survival/drug effects , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/drug effects , Drug Synergism , Female , Genetic Vectors/genetics , Genetic Vectors/metabolism , In Situ Nick-End Labeling , Male , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/genetics , Membrane Glycoproteins/pharmacology , Neural Cell Adhesion Molecules/biosynthesis , Neural Cell Adhesion Molecules/genetics , Neural Cell Adhesion Molecules/pharmacology , Neurons/cytology , Neurons/drug effects , Rats , Rats, Wistar
16.
J Biol Chem ; 277(32): 28861-9, 2002 Aug 09.
Article in English | MEDLINE | ID: mdl-12034732

ABSTRACT

We examined the possibility that basic fibroblast growth factor (bFGF) is involved in synaptic transmissions. We found that bFGF rapidly induced the release of glutamate and an increase in the intracellular Ca2+ concentration through voltage-dependent Ca2+ channels in cultured cerebral cortical neurons. bFGF also evoked a significant influx of Na+. Tetanustoxin inhibited the bFGF-induced glutamate release, revealing that bFGF triggered exocytosis. The mitogen-activated protein kinase (MAPK) pathway was required for these acute effects of bFGF. We also found that pretreatment with bFGF significantly enhanced high K+-elicited glutamate release also in a MAPK activation-dependent manner. Therefore, we propose that bFGF exerts promoting effects on excitatory neuronal transmission via activation of the MAPK pathway.


Subject(s)
Calcium/metabolism , Fibroblast Growth Factor 2/metabolism , Glutamic Acid/metabolism , Neurons/metabolism , Amino Acids/metabolism , Animals , Cell Division , Dose-Response Relationship, Drug , Enzyme Activation , Exocytosis , Fibroblast Growth Factors/metabolism , Immunoblotting , Immunohistochemistry , MAP Kinase Signaling System , Microscopy, Fluorescence , Potassium/metabolism , Rats , Signal Transduction , Sodium/metabolism , Tetanus Toxin/metabolism , Time Factors
17.
J Biol Chem ; 277(8): 6520-9, 2002 Feb 22.
Article in English | MEDLINE | ID: mdl-11741947

ABSTRACT

Brain-derived neurotrophic factor (BDNF) has been reported to exert an acute potentiation of synaptic activity. Here we examined the action of BDNF on synchronous spontaneous Ca(2+) oscillations in cultured cerebral cortical neurons prepared from postnatal 2-3-day-old rats. The synchronous spontaneous Ca(2+) oscillations began at approximately DIV 5. It was revealed that voltage-dependent Ca(2+) channels and ionotropic glutamate receptors were involved in the synchronous spontaneous oscillatory activity. BDNF potentiated the frequency of these oscillations. The BDNF-potentiated activity reached 207 +/- 20.1% of basal oscillatory activity. NT-3 and NT-4/5 also induced the potentiation. However, nerve growth factor did not. We examined the correlation between BDNF-induced glutamate release and the BDNF-potentiated oscillatory activity. Both up-regulation of phospholipase C-gamma (PLC-gamma) expression and the BDNF-induced glutamate release occurred at approximately DIV 5 when the BDNF-potentiated oscillations appeared. We confirmed that the BDNF-induced glutamate release occurred through a glutamate transporter that was dependent on the PLC-gamma/IP(3)/Ca(2+) pathway. Transporter inhibitors blocked the BDNF-potentiated oscillations, demonstrating that BDNF enhanced the glutamatergic transmissions in the developing cortical network by inducing glutamate release via a glutamate transporter.


Subject(s)
Brain-Derived Neurotrophic Factor/pharmacology , Calcium Signaling/drug effects , Cerebral Cortex/physiology , Neurons/drug effects , Amino Acid Transport System X-AG/metabolism , Animals , Animals, Newborn , Calcium Signaling/physiology , Cell Division/drug effects , Cells, Cultured , Cerebral Cortex/cytology , Glutamic Acid/metabolism , Glutamic Acid/pharmacology , Isoenzymes/metabolism , Kinetics , Mitogen-Activated Protein Kinases/metabolism , Neurons/cytology , Neurons/physiology , Neurotransmitter Agents/metabolism , Oncogene Proteins/metabolism , Oscillometry , Phosphatidylinositol 3-Kinases/metabolism , Phospholipase C gamma , Rats , Type C Phospholipases/metabolism
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