MAP1B binds to the NMDA receptor subunit NR3A and affects NR3A protein concentrations

Incorporation of the N-methyl-d-aspartate receptor (NMDAR) subunit NR3A into functional NMDARs results in reduced channel conductance and Ca²⁺ permeability. To further investigate the function of NR3A, we have set out to characterize its intracellular binding partners. Here, we report a novel protein interaction between NR3A and microtubule associated-protein (MAP) 1B, which both are localized to dendritic shafts and filopodia. NR3A protein levels were increased in MAP1B deficient (−/−) mice, with a corresponding decrease in NR1 levels, but the fraction of filopodia immunoreactive for NR3A was equal in cells from −/− and wild type (WT) mice. NR3A has previously been shown to interact with another member of the MAP1 family, MAP1S. We showed that MAP1S binds to microtubules in a similar manner as MAP1B, and suggest that MAP1S and MAP1B both are involved in regulating trafficking of NR3A-containing NMDAR.     

Influence of extremely low frequency magnetic fields on Ca signaling and NMDA receptor functions in rat hippocampus

Extremely low frequency (ELF < 300 Hz) electromagnetic fields affect several neuronal activities including memory. Because ELF magnetic fields cause altered Ca²⁺ homeostasis in neural tissues, we examined their influence on Ca²⁺ signaling enzymes in hippocampus and related them with NMDA receptor functions. Hippocampal regions were obtained from brains of 21-day-old rats that were exposed for 90 days to 50 Hz magnetic fields at 50 and 100 μT intensities. In comparison to controls, ELF exposure caused increased intracellular Ca²⁺ levels concomitant with increased activities of Ca²⁺-dependent protein kinase C (PKC), cAMP-dependent protein kinase and calcineurin as well as decreased activity of Ca²⁺–calmodulin-dependent protein kinase in hippocampal regions. Simultaneous ligand-binding studies revealed decreased binding to N-methyl-d-aspartic acid (NMDA) receptors. The combined results suggest that perturbed neuronal functions caused by ELF exposure may involve altered Ca²⁺ signaling events contributing to aberrant NMDA receptor activities.     

Inhibitory effects of Group I metabotropic glutamate receptors antagonists on the expression of NMDA receptor NR1 subunit in morphine tolerant rats

N-Methyl-d-aspartate receptor (NMDAR) and Group I metabotropic glutamate receptors (mGluRs) are involved in the process of morphine tolerance. Previous studies have shown that Group I mGluRs can modulate NMDAR functions in the central nervous system. The aim of the present study was to examine the influence of Group I mGluRs antagonists on the expression of NMDA receptor NR1 subunit (NR1) in the rat spinal cord. Morphine tolerance was induced in rats by repeated administration of 10 μg morphine (intrathecal, i.t.) twice a day for 7 consecutive days. Tail flick test was used to assess the effect of Group I mGluRs antagonist, AIDA ((RS)-1-Aminoindan-1,5 dicarboxylic acid) or mGluR5 antagonist, MPEP (2-methyl-6-(phenylethynyl)pyridine) on morphine antinociceptive tolerance. The expression of NR1 was measured by immunofluorescence and Western blot. Behavioral tests revealed that both AIDA and MPEP attenuated the development of morphine tolerance. The expression of NR1 was upregulated in the dorsal horn of spinal cord after chronic morphine treatment. AIDA or MPEP co-administered with morphine attenuated morphine induced upregulation of NR1. These findings suggest that the development of morphine tolerance partly prevented by Group I mGluRs antagonists may due to its inhibitory effect on the expression of NR1 subunit.     

Kinetics and functional characterization of the glycine receptor α2 and α3 subunit

In recent studies one serine residue (Ser-346) within the protein-kinase-A (R-E-S-R) consensus sequence of the GlyRα3 intracellular loop has proven to be an essential target for prostaglandin-E₂-mediated phosphorylation, which further modulates spinal nociceptive transmission and central inflammatory pain sensitization. In the present study we investigated the effect of Ser-346 phosphorylation and Ser-346 mutation on receptor kinetics and function using whole-cell patch-clamp recordings in transfected HEK 293 T cells. We compared biophysical properties of wild type GlyRα3 and two site-directed mutants, where Ser-346 was replaced by alanine or aspartate, in the absence and presence of prostaglandin-E₂. The mutation to alanine was accompanied by significantly altered dose–response and desensitization properties. Mutation to aspartate had only minor effects on receptor kinetics and function. Phosphorylation of Ser-346 slowed desensitization and decreased glycinergic currents in GlyRα3/mEP2 transfected cells. In addition, we demonstrated that prostaglandin-E₂ also had an effect on the GlyRα2 subunit. Exposure to prostaglandin-E₂ decreased the maximum peak current amplitude of glycinergic currents in GlyRα2/mEP2 transfected cells in the same manner as phosphorylation of the GlyRα3 subunit. It led to a significant increase of the desensitization time constants and thus significantly affected the desensitization behaviour. These results indicate that the GlyRα2 and the GlyRα3 subunits act as important subunits for the modulation of glycine receptor kinetics and function.     

Glycine and glycine receptor signaling in hippocampal neurons: Diversity,function and regulation

Glycine is a primary inhibitory neurotransmitter in the spinal cord and brainstem. It acts at glycine receptor (GlyR)-chloride channels, as well as a co-agonist of NMDA receptors (NMDARs). In the hippocampus, the study of GlyRs has largely been under-appreciated due to the apparent absence of glycinergic synaptic transmission. Emerging evidence has shown the presence of extrasynaptic GlyRs in the hippocampus, which exert a tonic inhibitory role, and can be highly regulated under many pathophysiological conditions. On the other hand, besides d-serine, glycine has also been shown to modulate NMDAR function in the hippocampus. The simultaneous activation of excitatory NMDARs and inhibitory GlyRs may provide a homeostatic regulation of hippocampal network function. Furthermore, glycine can regulate hippocampal neuronal activity through GlyR-mediated cross-inhibition of GABAergic inhibition, or through the glycine binding site-dependent internalization of NMDARs. Therefore, hippocampal glycine and its receptors may operate in concert to finely regulate hippocampus-dependent high brain function such as learning and memory. Finally, dysfunction of hippocampal glycine signaling is associated with neuropsychiatric disorders. We speculate that further studies of hippocampal glycine-mediated regulation may help develop novel glycine-based approaches for therapeutic developments.     

Src family kinase potentiates the activity of nicotinic acetylcholine receptor in rat autonomic ganglion innervating urinary bladder

Src family kinases (SFKs), one of the tyrosine kinase groups, are primary regulators of signal transductions that control cellular functions such as cell proliferation, differentiation, survival, metabolism, and other important roles of the cell. One of the crucial functions of SFKs is to regulate the activities of various neuronal channels. In this study, we investigated the modulatory action of SFK on nicotinic acetylcholine receptors (nAChRs) expressed in rat major pelvic ganglion (MPG) neurons innervating the urinary bladder. PP1 and PP2 (5 μM), selective Src-kinase inhibitors, attenuated ACh-induced ionic currents and [Ca2+](i) transients in MPG neurons, whereas PP3, an inactive analogue, had no effect. Blocking the tyrosine kinase activity of Src kinase by pp60 c-src inhibitory peptide also reduced the ACh-induced currents. Conversely, sodium orthovanadate (200 μM), a tyrosine phosphatase inhibitor, significantly augmented the ACh-induced currents. In the kinase assay, the activities of SFKs in MPG neurons were also inhibited by PP2, but not by PP3. These data suggests that SFKs may have a facilitative role on the synaptic transmission in rat pelvic autonomic ganglion.     

TrkB inhibition as a therapeutic target for CNS-related disorders

The interaction of brain-derived neurotrophic factor (BDNF) with its tropomyosin-related kinase receptor B (TrkB) is involved in fundamental cellular processes including neuronal proliferation, differentiation and survival as well as neurotransmitter release and synaptic plasticity. TrkB signaling has been widely associated with beneficial, trophic effects and many commonly used psychotropic drugs aim to increase BDNF levels in the brain. However, it is likely that a prolonged increased TrkB activation is observed in many pathological conditions, which may underlie the development and course of clinical symptoms. Interestingly, genetic and pharmacological studies aiming at decreasing TrkB activation in rodent models mimicking human pathology have demonstrated a promising therapeutic landscape for TrkB inhibitors in the treatment of various diseases, e.g. central nervous system (CNS) disorders and several types of cancer. Up to date, only a few selective and potent TrkB inhibitors have been developed. As such, the use of crystallography and in silico approaches to model BDNF-TrkB interaction and to generate relevant pharmacophores represent powerful tools to develop novel compounds targeting the TrkB receptor.     

The involvement of NR4A1 and NR4A2 in the regulation of the luteal function in rats

The nuclear receptor 4A (NR4A) members play important roles in cellular proliferation, differentiation and apoptosis. The current study first evaluate the expression of ovarian NR4A1 during different luteal stages in rats. Immature rats aged 28 days were treated with sequential Pregnant mare serum gonadotropin (PMSG) (D -2) / human chorionic gonadotropin (hCG) (D 0) to induce pseudopregnancy. Serum progesterone (P₄) and ovarian expression of NR4A1 were detected by RIA and WB, respectively, at follicle stage (D 0), early (D 2), middle (D 7) and late (D 14 and D 20) luteal stages. To confirm the role of NR4A1 during the luteal regression, rats were treated with prostaglandin F_2α analog (PGF) for 0–8?h on D 7 to detect the expressions of NR4A1 and NR4A2. RIA result showed that serum P₄ reached highest level on D 7 and then declined. WB results showed that there were two types of NR4A1 (NR4A1-L and NR4A1-S) expressed in the ovary. The ovarian NR4A1-L decreased at the late luteal stage (D 20). However, the NR4A1-S increased at the late luteal stage (D 14). After PGF treatment on D 7, the expression of NR4A1-S increased which peaked at 0.5–1?h and then declined; while NR4A1-L expression did not change within 8 h. Real-time PCR results showed that the ovarian NR4A1 mRNA increased within 0.5?h, maintained high at 1 h and then declined. The NR4A2 mRNA expression exhibited a similar pattern to that of NR4A1 mRNA, though its abundance was not as high as NR4A1. IHC results revealed that NR4A1-L was expressed mainly in the cytoplasm of luteal steroidogenic cells, faintly expressed in the follicle theca cells, oocytes and the pericytes; while NR4A2 was primarily localized in the cytoplasm of luteal steroidogenic cells. In conclusion, all these results demonstrate that NR4A2 as well as NR4A1 might be involved in the luteal development and luteolysis in rats.     

Roles of the ribosomal protein S19 dimer and the C5a receptor in pathophysiological functions of phagocytic leukocytes

Monocytes and neutrophils, the major phagocytic leukocytes, migrate to inflammatory sites by sensing chemoattractants such as anaphylatoxin C5a with membrane receptors such as C5a receptor. Upon stimulation, the leukocytes increase cytoplasmic Ca(2+) concentration and generate radical oxygen species. These leukocytes have different functions in inflammation. Neutrophils migrate more rapidly and induce vascular plasma leakage upon infiltration. Monocytes infiltrate tissue more slowly but have superior capacities of phagocytosis and antigen presentation. There must be mechanisms to separately recruit the leukocyte species at an inflammatory site. Ribosomal protein S19 (RP S19) is a component of ribosome. During apoptosis, RP S19 is dimerized and obtains a ligand capacity to C5a receptor. The RP S19 dimer attracts monocytes to phagocytically clear the apoptotic cells that released the dimer molecules. The phagocytic monocytes/macrophages then translocate to regional lymph nodes and present apoptotic cell-derived antigens. Oppositely, the RP S19 dimer inhibits C5a-induced neutrophil migration and promotes apoptosis of neutrophils via the C5a receptor. The RP S19 dimer seems to prevent excessive tissue destruction induced by neutrophils. Skp is a molecular chaperon of Gram-negative bacteria. Skp also attracts monocytes and neutrophils as a ligand of C5a receptor. However, it promotes neither cytoplasmic Ca(2+) enhancement nor radical oxygen generation.     

Evidence that Ca/calmodulin-dependent protein kinase mediates the modulation of the Ca2+-dependent K+ current,I AHP,by acetylcholine,but not by glutamate,in hippocampal neurons

Muscarinic and metabotropic glutamate receptor agonists increase the excitability of hippocampal and other cortical neurons by suppressing the Ca²⁺-activated K⁺current,I _AHP, which underlies the slow afterhyperpolarization (AHP) and spike frequency adaptation. We have examined the mechanism of action of a muscarinic agonist (carbachol) and a metabotropic glutamate receptor agonist (1-Aminocyclopentane-trans-1,3-dicarboxylic acid; t-ACPD) onI _AHP in hippocampal CA1 neurons in slices, by using highly specific protein kinase inhibitors. We found that inhibition of protein kinase A (PKA) with the adenosine 3,5-cyclic monophosphate (cAMP) analogue Rp-adenosine-3,5-cyclic phosphorothioate Rp-cAMPS, did not prevent the muscarinic and glutamatergic suppression ofI _AHP. In contrast, two specific peptide inhibitors of Ca²⁺/calmodulin-dependent protein kinase II (CaM-K II), each partially blocked the effect of carbachol, but not the effect of t-ACPD onI _AHP. We conclude that CaM-K II, but not PKA, is involved in mediating the muscarinic suppression ofI _AHP, although other pathways may also contribute. In contrast, neither CaM-K II nor PKA seems to mediate the metabotropic glutamate receptor action onI _AHP.     

Bone marrow-derived mesenchymal stem cells prevent the apoptosis of neuron-like PC12 cells via erythropoietin expression

Hippocampal cholinergic neurostimulating peptide (HCNP) induces the synthesis of acetylcholine in medial septal nucleus in vitro and in vivo. HCNP precursor protein (HCNP-pp) is a multifunctional protein that participates in a number of signaling pathways, including MAPK/extracellular signal and G-protein-coupled receptor kinase 2. We recently demonstrated that the amount of collapsin response mediator protein-2 (CRMP-2) is increased in hippocampus of HCNP-pp transgenic mice. To clarify the interaction between HCNP/HCNP-pp and CRMP-2 and its role in synaptic function, we investigated whether HCNP-pp is localized to the synapse and if it affects protein expression. Here, we demonstrate that HCNP-pp co-localizes with CRMP-2 at presynaptic terminals. Furthermore, HCNP-pp overexpression increases synaptophysin levels. These findings suggest that HCNP-pp, in association with CRMP-2, plays an important role in presynaptic function in the hippocampus.     

Mechanisms underlying the impaired regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in aged rat liver

As the main risk factor for cardiovascular disease, hypercholesterolemia is one of the most studied age-related metabolic alterations. In the liver, cholesterol homeostasis is strictly regulated through the modulation of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), the key enzyme of cholesterol biosynthesis. With ageing, hepatic HMG-CoA reductase becomes completely activated and cholesterol content increases in the blood. The research reported in this paper uses the regulatory enzymes of reductase (i.e., the AMP-dependent kinase (AMPK) and the protein phosphatase 2A (PP2A)), the HMG-CoA reductase thermodependent activity and the "in vitro" enzyme degradation to elucidate the role played by the HMG-CoA reductase regulation and its membrane interaction. Related experiments were performed on 3 and 24 months "ad libitum" (AL) fed rats and 24 months caloric-restricted rats. The results show no changes in the PP2A level and the activation state of AMP dependent kinase in aged "ad libitum" fed rats. By contrast, the activation state of the kinase is enhanced in the aged caloric-restricted animals. With respect to the adult, the thermodependent activity of reductase remains unchanged, while the degradation rate of the HMG-CoA reductase is slower and independent on proteasome. These findings support the hypothesis that a different arrangement of the HMG-CoA reductase membrane domain in aged rats is a cause of reductase deregulation.     

Differential distribution of MAP1A isoforms in the adult mouse barrel cortex and comparison with the serotonin 5-HT2A receptor

Microtubule-associated protein 1A (MAP1A) is essential during the late differentiation phase of neuronal development. Here, we demonstrated the presence of two MAP1A isoforms with a differential spatial distribution in the adult mouse barrel cortex. Antibody A stained MAP1A in pyramidal and stellate cells, including dendrites that crossed layer IV in the septa between barrels. The other antibody, BW6 recognized a MAP1A isoform that was mainly confined to the barrel hollow and identified smaller caliber dendrites. Previously, an interaction of MAP1A and the serotonin 5-hydroxytryptamine 2A (5-HT(2A)) receptor was shown in the rat cortex. Here, we identified, by double-immunofluorescent labeling, MAP1A isoform and serotonin 5-HT(2A) receptor distribution. MAP1A co-localized mainly with 5-HT(2A) receptor in larger apical dendrites situated in septa. This differential staining of MAP1A and a serotonin receptor in defined barrel compartments may be due to changes in the expression or processing of MAP1A during dendritic transport as a consequence of functional differences in processing of whisker-related sensory input.     

人参皂甙-Rd对SNI大鼠痛觉异常及脊髓背角内谷氨酸和NR2A/B表达的影响

目的:探讨人参皂甙-Rd(G-Rd)对大鼠神经病理性痛的镇痛效果及其机制。方法:成年雄性SD大鼠(30只)随机分成五组:空白对照组(blank control)、坐骨神经分支选择损伤组(spared nerve injury,SNI)、假手术组(sham operation)、SNI+saline(腹腔注射,i.p.)组、SNI+G-Rd(i.p.)组。行为学用von Frey法测定上述各组手术侧后肢机械缩足反射阈值(PWMT),以评定大鼠SNI术后痛敏变化以及人参皂甙-Rd的镇痛效果;用免疫荧光法检测对比上述各组大鼠脊髓L4-6节段背角内谷氨酸样和N-甲基-D-天冬氨酸受体2A/B亚单位(NR2A/B)样免疫阳性物的平均荧光强度(MFI)。结果:SNI术后10 d,手术侧PWMT值明显低于空白对照组和假手术组,术后20 d达到最低值,SNI+G-Rd组PWMT值明显高于SNI+Saline组(P<0.05)。免疫荧光染色显示SNI术后20 d,脊髓L4-6节段手术侧背角内谷氨酸样和NR2A/B样免疫阳性产物的MFI明显高于空白对照组和假手术组(P<0.05);SNI+G-Rd组脊髓L4-6节段手术侧背角内的谷氨酸样免疫阳性产物的MFI明显低于SNI和SNI+S组(P<0.05),而NR2A/B的MFI未见显著变化(P>0.05)。结论:人参皂甙-Rd可显著改善SNI引起的大鼠痛过敏行为,其可能的脊髓机制之一是与其有效地减少相应脊髓节段背角内谷氨酸的含量有关。     

GPCR mediated regulation of synaptic transmission

Synaptic transmission is a finely regulated mechanism of neuronal communication. The release of neurotransmitter at the synapse is not only the reflection of membrane depolarization events, but rather, is the summation of interactions between ion channels, G protein coupled receptors, second messengers, and the exocytotic machinery itself which exposes the components within a synaptic vesicle to the synaptic cleft. The focus of this review is to explore the role of G protein signaling as it relates to neurotransmission, as well as to discuss the recently determined inhibitory mechanism of Gβγ dimers acting directly on the exocytotic machinery proteins to inhibit neurotransmitter release.     

Significant linkage and association between a functional (GT)n polymorphism in promoter of the N-methyl-D-aspartate receptor subunit gene (GRIN2A) and schizophrenia

Dysfunction of the N-methyl-d-aspartate (NMDA) type glutamate receptor has been proposed as a mechanism in the etiology of schizophrenia, based on the observation that non-competitive antagonists of the NMDA receptor, such as phencyclidine, induce schizophrenia-like symptoms. Previous study identified a variable (GT)n polymorphism in the promoter region of the N-methyl-d-aspartate (NMDA) subunit gene (GRIN2A), and showed its association with schizophrenia in a case-control study, together with a correlation between the length of the repeat and severity of chronic outcome. Our present study was aimed at confirming the association of the (GT)n polymorphism of GRIN2A promoter with schizophrenia using 122 Han Chinese sib-pair families. Non-parametric linkage analysis and transmission/disequilibrium test (TDT) were undertaken using the GENEHUNTER, v2.1. In non-parametric linkage analysis, suggestive linkage was found for the (GT)n polymorphism (NPL=2.77, P=0.002902). The TDT was significant for (GT)n polymorphism and that the (GT)23 was preferentially transmitted to schizophrenia-affected children (T/NT: 123:72, chi(2)=13.34, P=0.000260). Our results indicate that the (GT)n polymorphism in the promoter of GRIN2A gene may play a significant role in the etiology of schizophrenia among our samples.