Differential regulation of cellular maturation in chondrocytes and osteoblasts by glycine

Previous studies have demonstrated the functional expression, by osteoblasts, of N-methyl-D-aspartate (NMDA) receptors responsible for the promotion of cellular differentiation in bone. We have now evaluated the possible role of the endogenous co-agonist of NMDA receptors, glycine (Gly), in chondrogenesis. In ex vivo organotypic cultures of fetal mouse tibias, proximal and distal cartilaginous primordia were significantly increased in the presence of Gly, with the osteogenic center being unchanged. Exposure to Gly drastically increased mRNA expression of the calcified chondrocyte marker osteopontin, without markedly affecting that of a proliferating chondrocyte marker or a hypertrophic chondrocyte marker, as shown in organotypic cultures by in situ hybridization analysis. Gly significantly increased Ca(2+) accumulation, osteopontin mRNA expression, and alkaline phosphatase activity in cultured rat costal chondrocytes, without significantly affecting those in cultured rat calvarial osteoblasts. The increase induced by Gly was significantly prevented by an NMDA receptor channel blocker and an antagonist at the Gly site on NMDA receptors, but not by an inhibitory Gly receptor antagonist or a Gly transporter inhibitor, in cultured chondrocytes. Constitutive mRNA expression was seen for NR1, NR2D, and NR3A subunits of NMDA receptors, but not for Gly receptors and transporters, in cultured chondrocytes. Corresponding immunoreactive proteins were detected for NR1 and NR2D subunits in cartilaginous zones of fetal mouse tibias. Thus, Gly might, at least in part, play a role as a trophic factor in the mechanisms associated with chondral calcification through the Gly site of NMDA receptors functionally expressed by chondrocytes in rodent cartilage.     

Developmental expression of NMDA receptors in primary cortical cell culture: relationship to function and excitotoxicity

Over‐activation of the N‐methyl‐d ‐aspartate (NMDA) receptor results in a Ca²⁺‐dependent neurotoxicity termed excitotoxicity. Primary neuronal cell cultures are often used to study the mechanisms of excitotoxicity. While the expression of the NMDA receptor (NR) subunits and their relationship to Ca²⁺ entry/accumulation and excitotoxicity has been studied extensively, all three parameters have not been examined concurrently. To determine unequivocally whether developmental expression of NR protein and function do indeed coincide with the appearance of excitotoxicity, we examined the temporal relationship between NR subunit expression, NMDA‐induced Ca²⁺ accumulation, and NMDA‐mediated excitotoxicity simultaneously using sister plates derived from the same mixed cortical cell culture preparations. Western Blot analysis of total protein isolated from cells cultured for 1, 4, 7, 10 and 14 days revealed a time‐dependent increase in NR1, NR2A and NR2B subunit expression, which surprisingly did not correlate with NMDA receptor function, as assessed by measurement of NMDA‐induced ⁴⁵Ca²⁺ accumulation. However, when only NR subunit surface expression was quantified, a correlation between expression and ⁴⁵Ca²⁺ accumulation did indeed exist. To our surprise, the emergence of excitotoxicity did not show a direct relationship to ⁴⁵Ca²⁺ accumulation as has been reported previously. Thus, it appears that other factors besides total Ca²⁺ accumulation must contribute to the emergence of excitotoxicity in mixed murine cortical cell cultures. Acknowledgements: Supported by a grant from The Patrick and Catherine Weldon Donaghue Medical Foundation.     

Structure of the zinc‐bound amino‐terminal domain of the NMDA receptor NR2B subunit

N‐methyl‐D ‐aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of fast excitatory synaptic transmission in the mammalian brain. One of the hallmarks for the function of NMDA receptors is that their ion channel activity is allosterically regulated by binding of modulator compounds to the extracellular amino‐terminal domain (ATD) distinct from the L ‐glutamate‐binding domain. The molecular basis for the ATD‐mediated allosteric regulation has been enigmatic because of a complete lack of structural information on NMDA receptor ATDs. Here, we report the crystal structures of ATD from the NR2B NMDA receptor subunit in the zinc‐free and zinc‐bound states. The structures reveal the overall clamshell‐like architecture distinct from the non‐NMDA receptor ATDs and molecular determinants for the zinc‐binding site, ion‐binding sites, and the architecture of the putative phenylethanolamine‐binding site.     

Amyloid precursor protein 695 associates with assembled NR2A‐ and NR2B‐containing NMDA receptors to result in the enhancement of their cell surface delivery

This is a study of the interaction between the two NMDA neurotransmitter receptor subtypes, NR1/NR2A and NR1/NR2B, and amyloid precursor protein (APP) 695, the major APP variant expressed in neurones. APP695 co‐immunoprecipitated with assembled NR1‐1a/NR2A and NR1‐1a/NR2B NMDA receptors following expression in mammalian cells. Single NR1‐1a, NR1‐2a, NR1‐4b^c‐Myc, or NR2 subunit transfections revealed that co‐association of APP695 with assembled NMDA receptors was mediated via the NR1 subunit; it was independent of the NR1 C1, C2, and C2′ cassettes and, the use of an NR1‐2a^c‐Myc‐trafficking mutant suggested that interaction between the two proteins occurs in the endoplasmic reticulum. The use of antibodies directed against extracellular and intracellular NR2 subunit epitopes for immunoprecipitations suggested that APP/NMDA receptor association was mediated via N‐terminal domains. Anti‐APP antibodies immunoprecipitated NR1, NR2A, and NR2B immunoreactive bands from detergent extracts of mammalian brain; reciprocally, anti‐NR1 or anti‐NR2A antibodies co‐immunoprecipitated APP immunoreactivity. Immune pellets from brain were sensitive to endoglycosidase H suggesting that, as for heterologous expression, APP and NMDA receptor association occurs in the endoplasmic reticulum. Co‐expression of APP695 in mammalian cells resulted in enhanced cell surface expression of both NR1‐1a/NR2A and NR1‐1a/NR2B NMDA receptors with no increase in total subunit expression. These findings are further evidence for a role of APP in intracellular trafficking mechanisms. Further, they provide a link between two major brain proteins that have both been implicated in Alzheimer’s disease.     

Overactivation of NR2B‐containing NMDA receptors through entorhinal–hippocampal connection initiates accumulation of hyperphosphorylated tau in rat hippocampus after transient middle cerebral artery occlusion

Middle cerebral artery occlusion (MCAO) induces secondary damages in the hippocampus that is remote from primary ischemic regions. Tau hyperphosphorylation is an important risk for neurodegenerative diseases. Increased tau phosphorylation has been identified in ischemic cortex, but little is known regarding the changes in the hippocampus. We showed that unilateral transient MCAO induced accumulation of hyperphosphorylated tau and concurrent dephosphorylation of glycogen synthase kinase‐3β at Ser 9 in the ipsilateral hippocampus. These MCAO‐induced changes were not reproduced when glutamatergic inputs from the entorhinal cortex to the hippocampus were transected; however, the changes were mimicked by intrahippocampal N‐methyl‐d ‐aspartate (NMDA) administration. Inhibition of NMDA receptor (NMDAR) subunit NR2B, but not NR2A activity in the hippocampus attenuated the accumulation of hyperphosphorylated tau and spatial cognitive impairment in MCAO rats. Together, our data suggest that overactivation of NR2B‐containing NMDARs through entorhinal–hippocampal connection plays an important role in the accumulation of hyperphosphorylated tau in the hippocampus following MCAO. Glycogen synthase kinase‐3β is an important protein kinase involved in NMDARs‐mediated tau hyperphosphorylation. This study indicates that early inhibition of NR2B‐containing NMDARs may represent a potential strategy to prevent or delay the occurrence of post‐stroke dementia.

     

Prostaglandin E2 stimulates glutamate receptor‐dependent astrocyte neuromodulation in cultured hippocampal cells

Recent Ca²⁺ imaging studies in cell culture and in situ have shown that Ca²⁺ elevations in astrocytes stimulate glutamate release and increase neuronal Ca²⁺ levels, and that this astrocyte‐neuron signaling can be stimulated by prostaglandin E₂ (PGE₂). We investigated the electrophysiological consequences of the PGE₂‐mediated astrocyte‐neuron signaling using whole‐cell recordings on cultured rat hippocampal cells. Focal application of PGE₂ to astrocytes evoked a Ca²⁺ elevation in the stimulated cell by mobilizing internal Ca²⁺ stores, which further propagated as a Ca²⁺ wave to neighboring astrocytes. Whole‐cell recordings from neurons revealed that PGE₂ evoked a slow inward current in neurons adjacent to astrocytes. This neuronal response required the presence of an astrocyte Ca²⁺ wave and was mediated through both N‐methyl‐D ‐aspartate (NMDA) and non‐NMDA glutamate receptors. Taken together with previous studies, these data demonstrate that PGE₂‐evoked Ca²⁺ elevations in astrocyte cause the release of glutamate which activates neuronal ionotropic receptors. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 221–229, 1999     

Influence of Mannosylation on Immunostimulating Activity of Adamant‐1‐yl Tripeptide

The mannosylated derivative of adamant‐1‐yl tripeptide (D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) was prepared to study the effects of mannosylation on adjuvant (immunostimulating) activity. Mannosylated adamant‐1‐yl tripeptide (Man‐OCH₂CH(Me)CO‐D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) is a non‐pyrogenic, H₂O‐soluble, and non‐toxic compound. Adjuvant activity of mannosylated adamantyl tripeptide was tested in the mouse model with ovalbumin as an antigen and in comparison to the parent tripeptide and peptidoglycan monomer (PGM, β‐D ‐GlcNAc‐(1→4)‐D ‐MurNAc‐L ‐Ala‐D ‐isoGln‐mesoDAP(εNH₂)‐D ‐Ala‐D ‐Ala), a well‐known effective adjuvant. The mannosylation of adamantyl tripeptide caused the amplification of its immunostimulating activity in such a way that it was comparable to that of PGM.     

Inflammatory cytokine‐mediated induction of serine racemase in atopic dermatitis

Serine racemase (SR) is an enzyme that catalyses the synthesis of d ‐serine, an endogenous coagonist for N‐methyl‐D‐aspartate (NMDA)‐type glutamate receptor in the central nervous system. Our previous study demonstrated that SR was expressed in the epidermis of wild‐type (WT) mice but not in SR knockout (KO) mice. In addition, SR immune‐reactivity was only found in the granular and cornified layers of the epidermis in WT mice. These findings suggested that SR is involved in the differentiation of epidermal keratinocytes and the formation of the skin barrier. However, its role in skin barrier dysfunction such as atopic dermatitis (AD) remains elusive. AD is a chronic inflammatory disease of skin, and the clinical presentation of AD has been reported to be occasionally associated with psychological factors. Therefore, this study examined the content of d ‐serine in stratum corneum in AD patients and healthy controls using a tape‐stripping method. Skin samples were collected from the cheek and upper arm skin of AD patient's lesion and healthy individuals. The d ‐serine content was significantly increased in the involved skin of AD in comparison with healthy individuals. An immunohistochemical analysis also revealed an increased SR expression in the epidermis of AD patients. Furthermore, the SR expression in cultured human keratinocytes was significantly increased by the stimulation with tumour necrosis factor ‐α or macrophage migration inhibitory factor. Taken together, these findings suggest that d ‐serine expressed particularly strongly in AD lesional skin and that the SR expression in the keratinocytes is linked to inflammatory cytokines.     

Synthesis and Immunostimulating Properties of Novel Adamant‐1‐yl Tripeptides

The aim of this work was to prepare L ‐ and D ‐(adamant‐1‐yl)‐Gly‐L ‐Ala‐D ‐isoGln peptides in order to study their adjuvant (immunostimulating) activities. Adjuvant activity of adamant‐1‐yl tripeptides was tested in the mouse model using ovalbumin as an antigen and in comparison to the peptidoglycan monomer (PGM; β‐D ‐GlcNAc‐(1→4)‐D ‐MurNAc‐L ‐Ala‐D ‐isoGln‐mesoDAP(εNH₂)‐D ‐Ala‐D ‐Ala) and structurally related adamant‐2‐yl tripeptides.     

NMDA neuroprotection against a phosphatidylinositol-3 kinase inhibitor, LY294002 by NR2B-mediated suppression of glycogen synthase kinase-3beta-induced apoptosis

To identify the intracellular signaling pathways that mediate the pro-survival activity of NMDA receptors (NMDARs), we studied effects of exogenous NMDA on cultured rat cortical and hippocampal neurons that were treated with a phosphatidylinositol-3-kinase (PI3K) inhibitor, LY294002. NMDA at 5 or 10 microm protected against LY294002-induced apoptosis, suggesting NMDAR-mediated activation of a survival signaling pathway that is PI3K-independent. NR2B-specific NMDAR blockers antagonized anti-apoptotic effects of NMDA, indicating a critical role of NR2B NMDARs in the neuroprotection. NMDA at 10 microm suppressed LY294002-induced activation of a pro-apoptotic kinase, glycogen synthase kinase 3beta (GSK3beta). GSK3beta activation by LY294002 was associated with decreased levels of inhibitory GSK3beta phosphorylation at the Ser9 residue. However, NMDA did not prevent the LY294002-mediated decline of phospho-Ser9 levels. In addition, NMDA inhibited cortical neuron apoptosis induced by the overexpression of either wild type (wt) or Ser9Ala mutant form of GSK3beta, suggesting that NMDA suppressed GSK3beta in a Ser9-independent manner. Finally, inhibition of NR2B NMDARs reduced the NMDA protection against overexpression of GSK3betawt. These data indicate that moderate stimulation of NR2B NMDAR protects against inhibition of PI3K by a Ser9-independent inhibition of the pro-apoptotic activity of GSK3beta. Hence, the activation of NR2B and the Ser9-independent inhibition of GSK3beta are two newly identified elements of the signaling network that mediates the pro-survival effects of NMDA.     

Relevance of weak flavin binding in human D‐amino acid oxidase

In the brain, the human flavoprotein D ‐amino acid oxidase (hDAAO) is involved in the degradation of the gliotransmitter D ‐serine, an important modulator of NMDA‐receptor‐mediated neurotransmission; an increase in hDAAO activity (that yields a decrease in D ‐serine concentration) was recently proposed to be among the molecular mechanisms leading to the onset of schizophrenia susceptibility. This human flavoenzyme is a stable homodimer (even in the apoprotein form) that distinguishes from known D ‐amino acid oxidases because it shows the weakest interaction with the flavin cofactor in the free form. Instead, cofactor binding is significantly tighter in the presence of an active site ligand. In order to understand how hDAAO activity is modulated, we investigated the FAD binding process to the apoprotein moiety and compared the folding and stability properties of the holoenzyme and the apoprotein forms. The apoprotein of hDAAO can be distinguished from the holoenzyme form by the more “open” tertiary structure, higher protein fluorescence, larger exposure of hydrophobic surfaces, and higher sensitivity to proteolysis. Interestingly, the FAD binding only slightly increases the stability of hDAAO to denaturation by urea or temperature. Taken together, these results indicate that the weak cofactor binding is not related to protein (de)stabilization or oligomerization (as instead observed for the homologous enzyme from yeast) but rather should represent a means of modulating the activity of hDAAO. We propose that the absence in vivo of an active site ligand/substrate weakens the cofactor binding, yielding the inactive apoprotein form and thus avoiding excessive D ‐serine degradation.     

Binding of spermine and ifenprodil to a purified,soluble regulatory domain of the N‐methyl‐d‐aspartate receptor

The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N‐methyl‐D ‐aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1‐R, NR2A‐R and NR2B‐R. The R domains were over‐expressed in Escherichia coli and purified to near homogeneity. The K_d values for binding of [¹⁴C]spermine to NR1‐R, NR2A‐R and NR2B‐R were 19, 140, and 33 μM, respectively. [³H]Ifenprodil bound to NR1‐R (K_d, 0.18 μM) and NR2B‐R (K_d, 0.21 μM), but not to NR2A‐R at the concentrations tested (0.1–0.8 μM). These K_d values were confirmed by circular dichroism measurements. The K_d values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1‐R and NR2A‐R or NR1‐R and NR2B‐R was additive with that of each individual R domain. Binding of spermine to NR1‐R and NR2B‐R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1‐R and NR2B‐R are distinct.     

Acute inhibitory effect of alpha‐mangostin on sarcoplasmic reticulum calcium‐ATPase and myocardial relaxation

The benefits of α‐mangostin for various tissues have been reported, but its effect on the heart has not been clarified. This study aimed to evaluate the effects of α‐mangostin on cardiac function. Using a cardiac sarcoplasmic reticulum (SR) membrane preparation, α‐mangostin inhibited SR Ca²⁺‐ATPase activity in a dose‐dependent manner (IC₅₀ of 6.47 ± 0.7 μM). Its suppressive effect was specific to SR Ca²⁺‐ATPase but not to myofibrillar Ca²⁺‐ATPase. Using isolated cardiomyocytes, 50 μM of α‐mangostin significantly increased the duration of cell relengthening and increased the duration of Ca²⁺ transient decay, suggesting altered myocyte relaxation. The relaxation effect of α‐mangostin was also supported in vivo after intravenous infusion. A significant suppression of both peak pressure and rate of ventricular relaxation (–dP/dt) relative to DMSO infusion was observed. The results from the present study demonstrated that α‐mangostin exerts specific inhibitory action on SR Ca²⁺‐ATPase activity, leading to myocardial relaxation dysfunction.     

Regulation of NR1/NR2C N-methyl-D-aspartate (NMDA) receptors by phosphorylation

NR2C-containing N-methyl-D-aspartate (NMDA) receptors are highly expressed in cerebellar granule cells where they mediate the majority of current in the adult. NMDA receptors composed of NR1/NR2C exhibit a low conductance and reduced sensitivity to Mg(2+), compared with the more commonly studied NR2A- and NR2B-containing receptors. Despite these interesting features, very little is known about the regulation of NR2C function. Here we investigate the role of phosphorylation of NR2C in regulating NMDA receptor trafficking and ion channel properties. We identify a phosphorylation site, serine 1244 (Ser(1244)), near the extreme COOH terminus of NR2C, which is phosphorylated by both cAMP-dependent protein kinase and protein kinase C. This residue is located adjacent to the consensus PDZ ligand, a region that regulates protein-protein interactions and receptor trafficking in NR2A and NR2B. We show that Ser(1244) on NR2C is phosphorylated in vitro, in heterologous cells, and in neurons. Moreover, we demonstrate for the first time that NR2C interacts with the PSD-95 family of PDZ domain-containing proteins but that phosphorylation of Ser(1244) does not influence this PDZ interaction. Furthermore, Ser(1244) phosphorylation does not regulate surface expression of NR1/NR2C receptors. However, we find that this site does regulate the kinetics of the ion channel: a phosphomimetic mutation at Ser(1244) accelerates both the rise and decay of NMDA-evoked currents in excised patches from HEK-293 cells. Therefore, phosphorylation of Ser(1244) does not regulate trafficking but unexpectedly affects ion channel function, suggesting that phosphorylation of Ser(1244) on NR2C may be important in defining the functional properties of NMDA receptor-mediated currents in the cerebellum.     

Sociability development in mice with cell‐specific deletion of the NMDA receptor NR1 subunit gene

Social affiliative behavior is an important component of everyday life in many species and is likely to be disrupted in disabling ways in various neurodevelopmental and neuropsychiatric disorders. Therefore, determining the mechanisms involved in these processes is crucial. A link between N‐methyl‐d ‐aspartate (NMDA) receptor function and social behaviors has been clearly established. The cell types in which NMDA receptors are critical for social affiliative behavior, however, remain unclear. Here, we use mice carrying a conditional allele of the NMDA R1 subunit to address this question. Mice bearing a floxed NMDAR1 (NR1) allele were crossed with transgenic calcium/calmodulin‐dependent kinase IIα (CaMKIIα)‐Cre mice or parvalbumin (PV)‐Cre mice targeting postnatal excitatory forebrain or PV‐expressing interneurons, respectively, and assessed using the three‐chambered Social Approach Test. We found that deletion of NR1 in PV‐positive interneurons had no effect on social sniffing, but deletion of NR1 in glutamatergic pyramidal cells resulted in a significant increase in social approach behavior, regardless of age or sex. Therefore, forebrain excitatory neurons expressing NR1 play an important role in regulating social affiliative behavior.     

The Tau/A152T mutation,a risk factor for frontotemporal‐spectrum disorders,leads to NR2B receptor‐mediated excitotoxicity

We report on a novel transgenic mouse model expressing human full‐length Tau with the Tau mutation A152T (hTau^AT), a risk factor for FTD‐spectrum disorders including PSP and CBD. Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis‐sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short‐ or long‐term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage‐gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau^AT causes excitotoxicity mediated by NR2B‐containing NMDA receptors due to enhanced extracellular glutamate.     

Runx2‐mediated bcl‐2 gene expression contributes to nitric oxide protection against hydrogen peroxide‐induced osteoblast apoptosis

Nitric oxide (NO) can regulate osteoblast activities. This study was aimed to evaluate the protective effects of pretreatment with sodium nitroprusside (SNP) as a source of NO on hydrogen peroxide‐induced osteoblast insults and its possible mechanisms. Exposure of human osteosarcoma MG63 cells to hydrogen peroxide significantly increased cellular oxidative stress, but decreased ALP activity and cell viability, inducing cell apoptosis. Pretreatment with 0.3 mM SNP significantly lowered hydrogen peroxide‐induced cell insults. Treatment of human MG63 cells with hydrogen peroxide inhibited Bcl‐2 mRNA and protein production, but pretreatment with 0.3 mM SNP significantly ameliorated such inhibition. Sequentially, hydrogen peroxide decreased the mitochondrial membrane potential, but increased the levels of cytochrome c and caspase‐3 activity. Pretreatment with 0.3 mM SNP significantly lowered such alterations. Exposure to hydrogen peroxide decreased Runx2 mRNA and protein syntheses. However, pretreatment with 0.3 mM SNP significantly lowered the suppressive effects. Runx2 knockdown using RNA interference inhibited Bcl‐2 mRNA production in human MG63 cells. Protection of pretreatment with 0.3 mM SNP against hydrogen peroxide‐induced alterations in ALP activity, caspase‐3 activity, apoptotic cells, and cell viability were also alleviated after administration of Runx2 small interference RNA. Thus, this study shows that pretreatment with 0.3 mM SNP can protect human MG63 cells from hydrogen peroxide‐induced apoptotic insults possibly via Runx2‐involved regulation of bcl‐2 gene expression. J. Cell. Biochem. 108: 1084–1093, 2009. © 2009 Wiley‐Liss, Inc.