bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of Ca2+/Calmodulin‐Dependent Protein Kinase II by Extracellular Calcium in Cultured Hippocampal Pyramidal Neurons
Authors: Scholz Wendy K. , Palfrey H. Clive
Published: July 2010

Abstract: The ability of various stimuli to convert Ca2+/calmodulin‐dependent protein kinase II (CaMKII) into a Ca2+‐independent (autonomous) form was examined in cultured embryonic rat hippocampal pyramidal neurons. The most effective stimulation by far was observed when cells were equilibrated in buffer containing low extracellular [Ca2+] ([Ca2+]o) (∼50 nM) and then shifted to normal [Ca2+]o (∼1.26 mM) by addition of CaCl2 (referred to as “Ca2+ stimulation”). Virtually complete (>90%) conversion of the kinase to the autonomous form occurred within 30–50 s, with a return to baseline within 5 min. By contrast, depolarization of cells with high [K+] or treatment with glutamate or a Ca2+ ionophore caused insignificant increases (<10%) in levels of the autonomous form. The Ca2+‐stimulated increase in CaMKII autonomy coincided with a two‐ to threefold increase in kinase subunit phosphorylation. In the first 40 s of Ca2+ stimulation, 32P incorporation into the immunoprecipitated subunits of CaMKII occurred exclusively on threonine residues, including Thr286Thr287 of the α/β subunits. Longer incubation of cells resulted in a decline of phosphothreonine content, whereas levels of phosphoserine‐containing peptides showed a significant increase. The activation of CaMKII by Ca2+ stimulation was accompanied by only a small rise in intracellular [Ca2+]. Inhibitor studies showed that Na+‐dependent action potentials and Ca2+ influx through glutamate receptors or voltage‐sensitive Ca2+ channels did not contribute to the activation. Moreover, CaMKII was not activated by extracellular addition of other cations, including Mn2+, Mg2+, Co2+, or Gd3+. Although the mechanism of Ca2+ stimulation is presently unclear, it may involve either activation of extracellular calcium receptors or capacitative calcium entry. The dramatic rise in CaMKII autonomy and the Ca2+ selectivity of the response suggest a direct and specific relationship between [Ca2+]o and the state of activation of the kinase in intact neurons.
Muscular Dystrophy in mdx Mice Despite Lack of Neuronal Nitric Oxide Synthase
Authors: Chao Daniel S. , Silvagno Francesca , Bredt David S.
Published: July 2010

Abstract: Neuronal nitric oxide synthase (nNOS) is a component of the dystrophin complex in skeletal muscle. The absence of dystrophin protein in Duchenne muscular dystrophy and in mdx mouse causes a redistribution of nNOS from the plasma membrane to the cytosol in muscle cells. Aberrant nNOS activity in the cytosol can induce free radical oxidation, which is toxic to myofibers. To test the hypothesis that derangements in nNOS disposition mediate muscle damage in Duchenne dystrophy, we bred dystrophin‐deficient mdx male mice and female mdx heterozygote mice that lack nNOS. We found that genetic deletion of nNOS does not itself cause detectable pathology and that removal of nNOS does not influence the extent of increased sarcolemmal permeability in dystrophin‐deficient mice. Thus, histological analyses of nNOS‐dystrophin double mutants show pathological changes similar to the dystrophin mutation alone. Taken together, nNOS defects alone do not produce muscular dystrophy in the mdx model.
Role of Caspases in N‐Methyl‐d‐Aspartate‐Induced Apoptosis in Cerebrocortical Neurons
Authors: Tenneti Lalitha , D'Emilia Danielle M. , Troy Carol M. , Lipton Stuart A.
Published: July 2010

Abstract: Overactivation of glutamate receptors mediates neuronal death in several acute and chronic neurodegenerative diseases. The intracellular processes underlying this form of death, however, remain poorly understood. Depending on the severity of insult, N‐methyl‐d‐aspartate (NMDA) receptor activation induces either apoptosis or necrosis. Cysteine proteases related to interleukin‐1β‐converting enzyme (ICE), recently termed caspases, appear necessary for neuronal apoptosis in vivo and in vitro. To determine whether caspases play a role in NMDA‐induced apoptosis, we used two functionally distinct approaches to decrease substrate cleavage by caspases. One is a novel peptide (V‐ICEinh) that contains the caspase catalytic site and acts as a pseudoenzyme that binds caspase substrates and prevents their cleavage. The other is a pseudosubstrate peptide (Z‐VAD·fmk) that inhibits caspase activity. Pretreatment with either V‐ICEinh or Z‐VAD·fmk protects cerebrocortical neurons from NMDA‐induced apoptosis, suggesting a role for caspases in NMDA‐induced apoptosis. To explore the signaling pathways involved, we looked at the effects of NMDA receptor activation on Ca2+ influx, production of reactive oxygen species (ROS), mitochondrial membrane potential, and lipid peroxidation. Neither NMDA‐induced Ca2+ influx nor the initial collapse of mitochondrial membrane potential could be prevented by pretreatment with V‐ICEinh or Z‐VAD·fmk. In contrast, ROS formation and lipid peroxidation were completely blocked by both V‐ICEinh and Z‐VAD·fmk. Taken together, our results suggest that Ca2+ influx and mitochondrial depolarization occur upstream from caspase activation, whereas ROS formation and lipid peroxidation may be downstream events in the cascade leading to cortical neuronal apoptosis.
The Cholinergic Gene Locus Is Coordinately Regulated by Protein Kinase A II in PC12 Cells
Authors: Shimojo Masahito , Wu Donghai , Hersh Louis B.
Published: July 2010

Abstract: The vesicular acetylcholine transporter (VAChT) gene and the choline acetyltransferase (ChAT) gene comprise the cholinergic gene locus. We have studied the coordinate regulation of these genes by cyclic AMP‐dependent protein kinase (PKA) in the rat pheochromocytoma cell line PC12 and PC12 PKA‐deficient mutants. Both ChAT and VAChT mRNA increased approximately fourfold after treatment of PC12 cells with dibutyryl cyclic AMP (dbcAMP). ChAT and PKA activity were also increased by dbcAMP. The basal levels of ChAT and VAChT mRNAs in the PKA‐deficient cell lines were both about six times lower than in wild‐type PC12 cells, and were induced less than twofold by addition of dbcAMP. H‐89 and H‐9, specific inhibitors for PKA, reduced ChAT and VAChT mRNA levels to approximately one‐third that of untreated cells and ChAT activity to approximately one‐fourth that of untreated PC12 cells. Activation of PKA type II, but not PKA type I, increased ChAT activity approximately threefold. Analysis of reporter gene constructs indicates that PKA affects gene transcription at an upstream site in the cholinergic gene locus. These results demonstrate that the expression of the ChAT and VAChT genes is regulated coordinately at the transcriptional level, and a signaling pathway specifically involving PKA II plays an important role in this process.
Antisense Oligonucleotide‐Induced Reduction in 5‐Hydroxytryptamine7 Receptors in the Rat Hypothalamus Without Alteration in Exploratory Behaviour or Neuroendocrine Function
Authors: Clemett D. A. , Cockett M. I. , Marsden C. A. , Fone K. C. F.
Published: July 2010

Abstract: The effect of a 5‐hydroxytryptamine7 (5‐HT7) receptor‐directed antisense oligonucleotide on rat behaviour and neuroendocrine function was investigated. Six days of intracerebroventricular 5‐HT7 antisense oligonucleotide treatment significantly reduced [3H]5‐HT binding to hypothalamic 5‐HT7 receptors, whereas cortical 5‐HT2C density remained unchanged. In rats on a food‐restricted diet, both antisense and mismatch oligonucleotides reduced food intake and body weight compared with that in vehicle‐treated controls by day 4 of administration. 5‐HT7 antisense oligonucleotide administration did not affect exploratory or locomotor activity in photocell activity monitors on day 4 or elevated plus‐maze behaviour on day 6 of intracerebroventricular treatment. 5‐HT7 antisense oligonucleotide did not affect plasma corticosterone or prolactin levels or 5‐HT turnover in either 5‐HT cell body or terminal areas. These data demonstrate that intracerebroventricular 5‐HT7 antisense oligonucleotide administration selectively reduced rat hypothalamic 5‐HT7 receptor density without affecting any of the biochemical or behavioural measures. The results suggest that this antisense protocol could be a valuable tool to investigate central 5‐HT7 receptor functions, and that this receptor is not critical for the control of neuroendocrine function or food intake.
Molecular Cloning and Characterization of the Rat P2Y4 Receptor
Authors: Webb Tania E. , Henderson Duncan J. , Roberts Jonathan A. , Barnard Eric A.
Published: July 2010

Abstract: Degenerate PCR was used to amplify DNAs encoding members of the P2Y receptor family from rat brain RNA. A full‐length sequence obtained for one novel clone (R5) contained an intronless open reading frame that encoded a polypeptide of 361 amino acids, sharing 84% sequence identity with the human P2Y4 receptor. When R5 was stably expressed in Jurkat cells, calcium fluxes resulting from stimulation of the receptor showed that UDP, ADP, 2‐methylthio‐ATP, and diadenosine tetraphosphate were inactive, whereas UTP and ATP were both full agonists with similar potency. At the human receptor, ATP has significantly lower potency than UTP. The R5 transcript was not detected in brain by northern hybridization. Therefore, its tissue distribution was assessed by PCR, and the mRNA was found to be widely distributed at a low abundance, being present in brain, spinal cord, and a variety of peripheral organs. Localization of the receptor transcript in adult rat brain sections by in situ hybridization indicated that it is expressed at highest levels in the pineal gland and ventricular system. It is presumed that R5 is a species orthologue of the human P2Y4 receptor but with this significant difference in agonist pharmacology.
Migration Behavior of Rodent Granule Neurons in the Presence of Antibody to the 4C5 Antigen
Authors: Yfanti Eleni , Nagata Isao , Patsavoudi Evangelia
Published: July 2010

Abstract: We have reported the production of monoclonal antibody 4C5, which recognizes a cell surface antigen, the 4C5 antigen, involved in granule cell migration processes. In the present study, we investigated in a more precise manner the role of the 4C5 antigen in the different types of granule cell migrations that take place during cerebellar development. When cerebellar explant cultures derived from 10‐day‐old rats were performed for 2 days in the presence of monoclonal antibody 4C5, vertical granule cell migration, occurring in the presence of glia, was not significantly inhibited. In contrast, when monoclonal antibody 4C5 was included in the medium of microexplant cultures derived from 4‐day‐old mice and maintained for 4 days in vitro, granule cell migrations that occurred both parallel and perpendicular to the neurite bundles that were free of glia were inhibited. Moreover, a stronger inhibitory effect of the antibody was observed on migration perpendicular to the neurite bundles compared with the parallel type of migration. Our results indicate that the 4C5 antigen differentially affects the different developmental stages and types of granule cell migration during rodent cerebellar development.
Butyrylcholinesterase Antisense Transfection Increases Apoptosis in Differentiating Retinal Reaggregates of the Chick Embryo
Authors: Robitzki Andrea , Mack Alexandra , Hoppe Ulrike , Chatonnet Arnaud , Layer Paul G.
Published: July 2010

Abstract: To investigate the roles of the enzymes butyryl‐ and acetylcholinesterase (BChE and AChE) in retinal proliferation and differentiation, we use reaggregated spheres from retinal cells of the 6‐day‐old chick embryo, forming cellular and fibrous areas homologous to all layers of a normal retina. Recently, we could suppress BChE expression by transfecting these so‐called retinospheroids during their proliferation period with a pSVK3 expression vector containing a 5′ fragment of the rabbit BChE gene in antisense orientation. Along with morphological changes, proliferation was significantly decreased. Here, we have studied the effect of antisense BChE suppression during the differentiation period of retinospheroids. As BChE is suppressed, the differentiation of AChE‐positive cells is increased, whereas the immunoreactivities for red and green cone‐specific opsins are strongly reduced. Concomitantly, the rate of apoptosis as determined by propidium iodide uptake, by increased CPP 32‐like caspase expression, and by terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling and DNA fragmentation assays is roughly doubled, predominantly at the expense of degenerating photoreceptor precursors. This is further strong evidence that the proliferation marker BChE regulates an intricate balance between cell proliferation, cell differentiation, and programmed cell death in this in vitro retinal system.
Cannabinoid Receptor Activation Differentially Regulates the Various Adenylyl Cyclase Isozymes
Authors: Rhee Man‐Hee , Bayewitch Michael , Avidor‐Reiss Tomer , Levy Rivka , Vogel Zvi
Published: July 2010

Abstract: Two cannabinoid receptors belonging to the superfamily of G protein‐coupled membrane receptors have been identified and cloned: the neuronal cannabinoid receptor (CB1) and the peripheral cannabinoid receptor (CB2). They have been shown to couple directly to the Gi/o subclass of G proteins and to mediate inhibition of adenylyl cyclase upon binding of a cannabinoid agonist. In several cases, however, cannabinoids have been reported to stimulate adenylyl cyclase activity, although the mechanism by which they did so was unclear. With the cloning of nine adenylyl cyclase isozymes with various properties, including different sensitivities to αs, αi/o, and βγ subunits, it became important to assess the signaling pattern mediated by each cannabinoid receptor via the different adenylyl cyclase isozymes. In this work, we present the results of cotransfection experiments between the two types of cannabinoid receptors and the nine adenylyl cyclase isoforms. We found that independently of the method used to stimulate specific adenylyl cyclase isozymes (e.g., ionomycin, forskolin, constitutively active αs, thyroid‐stimulating hormone receptor activation), activation of the cannabinoid receptors CB1 and CB2 inhibited the activity of adenylyl cyclase types I, V, VI, and VIII, whereas types II, IV, and VII were stimulated by cannabinoid receptor activation. The inhibition of adenylyl cyclase type III by cannabinoids was observed only when forskolin was used as stimulant. The activity of adenylyl cyclase type IX was inhibited only marginally by cannabinoids.
Vacuolar H+‐ATPase Domains Are Transported Separately in Axons and Assemble in Torpedo Nerve Endings
Authors: Morel Nicolas , Gérard Valérie , Shiff Gad
Published: July 2010

Abstract: Torpedo electric organ synaptosomes possess a typical vacuolar H+‐ATPase (V‐ATPase), inhibited by concanamycin A and insensitive to vanadate, made of the association of a catalytic soluble sector V1 to a membrane domain V0. In the electric nerves, the 57‐kDa subunit B of the V1 sector was transported to the nerve endings by the slow axonal flow and did not accumulate upstream from an axonal block. In contrast, a 500% accumulation of the 15‐kDa subunit c of the V0 membrane domain was observed, demonstrating that this subunit is conveyed by the fast axonal anterograde transport. After velocity sedimentation of solubilized nerve proteins, the 57‐ and 15‐kDa subunits were recovered in different complexes corresponding, respectively, to the V1 and V0 domains. No fully assembled V‐ATPase was detected. It is concluded that V1 and V0 domains of V‐ATPase are transported separately in axons, at different rates, and that they only associate once arrived in nerve endings to form the active V‐ATPase.
Calcium/Calmodulin‐Dependent Protein Kinase II Is Associated with NR2A/B Subunits of NMDA Receptor in Postsynaptic Densities
Authors: Gardoni F. , Caputi A. , Cimino M. , Pastorino L. , Cattabeni F. , Di Luca M.
Published: July 2010

Abstract: NMDA receptors and Ca2+/calmodulin‐dependent kinase II (CaMKII) have been reported to be highly concentrated in the postsynaptic density (PSD). Although the possibility that CaMKII in PSD might be associated with specific proteins has been put forward, the protein or proteins determining the targeting of the kinase in PSD have not yet been identified. Here we report that CaMKII binds to NR2A and NR2B subunits of NMDA receptors in PSD isolated from cortex and hippocampus. The association of NMDA receptor subunits and CaMKII was assessed by immunoprecipitating PSD proteins with antibodies specific for NR2A/B and CaMKII: CaMKII coprecipitated with NR2A/B and NR1 but not with other glutamate ionotropic receptor subunits, such as GluR1 and GluR2‐3. A direct association between CaMKII and NR2A/B subunits was further confirmed by overlay experiments using either 32P‐autophosphorylated CaMKII or 32P‐NR2A/B and by evaluating the formation of a CaMKII‐NR2A/B complex by means of the cross‐linker disuccimidyl suberate. These data demonstrate an association between the NMDA receptor complex and CaMKII in the postsynaptic compartment, suggesting that this colocalization may be relevant for synaptic plasticity.
A Molecular Model of Myelin Oligodendrocyte Glycoprotein
Authors: Hjelmström Peter , Penzotti Julie E. , Henne Randal M. , Lybrand Terry P.
Published: July 2010

Abstract: Myelin oligodendrocyte glycoprotein (MOG) is a protein on the surface of myelin sheaths. It is a putative target of the autoimmune attack in the inflammatory and demyelinating CNS disease multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. MOG belongs to the immunoglobulin superfamily (IgSF), and its extracellular N‐terminal domain contains many conserved IgSF consensus residues seen in immunoglobulin variable region folds. The aim of the present study was to create a molecular model of the extracellular N‐terminal domain of mouse MOG. No crystal structure is yet available of MOG, and thus a molecular model would be useful in providing insight into its structure and binding characteristics. Molecular graphics techniques and molecular dynamics with secondary structure‐based restraints were used in the construction and refinement of the MOG model. Regions of high prediction confidence were identified, and possible glycosylation, dimerization, complement binding, and antibody‐binding regions in MOG were mapped and analyzed.
Externalization of Phosphatidylserine May Not Be an Early Signal of Apoptosis in Neuronal Cells, but Only the Phosphatidylserine‐Displaying Apoptotic Cells Are Phagocytosed by Microglia
Authors: Adayev Tatyana , Estephan Rima , Meserole Susan , Mazza Bethanne , Yurkow Edward J. , Banerjee Probal
Published: July 2010

Abstract: Earlier reports on nonneural cells have shown that the normally inner plasma membrane lipid, phosphatidylserine (PS), flip‐flops out during the early stages of apoptosis, whereas DNA laddering and plasma membrane permeabilization occur during the late stages. In this study, the applicability of these parameters to CNS‐derived neuronal cells was tested using hippocampal HN2‐5, cells that undergo apoptosis under anoxia. Because such insults on unsynchronized cells, e.g., undifferentiated HN2‐5 cells, result in both early and late apoptotic cells, we mechanically separated these cells into three fractions containing (a) cells that had completely detached during anoxia, (b) cells that remained weakly attached to the tissue culture dish and, once detached by trituration in serum‐containing medium, did not reattach, and (c) cells that reattached in 2–3 h. Fractions a and b contained cells that showed pronounced DNA laddering, whereas cells in fraction c did not show any DNA laddering. Double staining with fluorescein isothiocyanate‐annexin V (which binds to PS) and propidium iodide (which stains the DNA in cells with a permeable cell membrane) revealed that all cells in fraction a had a permeable cell membrane (propidium iodide‐positive) and PS molecules in the outer leaflet of the plasma membrane (fluorescein isothiocyanate‐annexin V‐positive). By contrast, fractions b and c contained cells with no externalized PS molecules. Cells in fractions a–c also showed, respectively, 50‐, 21‐, and 5.5‐fold higher caspase‐3 (CPP32) activity than that in healthy control cells. All these results show that fraction a contained late apoptotic cells, which also had the highest CPP32 activity; cells in fraction b were at an intermediate stage, when DNA laddering had already occurred; and fraction c contained very early apoptotic cells, in which no DNA laddering had yet occurred. Therefore, in the neuronal HN2‐5 cells, externalization of PS occurs only during the final stages of apoptosis when the cells have completely lost their adhesion properties. Further experiments showed that ameboid microglial cells isolated from neonatal mouse brain phagocytosed only the cells in fraction a. These results show that in CNS‐derived HN2‐5 cells, (a) PS externalization is a late apoptotic event and is concomitant with a complete loss of surface adhesion of the apoptotic cells and (b) PS externalization is crucial for microglial recognition and phagocytosis of the apoptotic HN2‐5 cells. Thus, PS externalization could be causally linked to the final detachment of apoptotic neuronal cells, which in turn prepares them for rapid phagocytosis by microglia.
Generation and Regulation of β‐Amyloid Peptide Variants by Neurons
Authors: Gouras Gunnar K. , Xu Huaxi , Jovanovic Jasmina N. , Buxbaum Joseph D. , Wang Rong , Greengard Paul , Relkin Norman R. , Gandy Sam
Published: July 2010

Abstract: Studies of processing of the Alzheimer β‐amyloid precursor protein (βAPP) have been performed to date mostly in continuous cell lines and indicate the existence of two principal metabolic pathways: the “β‐secretase” pathway, which generates β‐amyloid (Aβ1–40/42; ∼4 kDa), and the “α‐secretase” pathway, which generates a smaller fragment, the “p3” peptide (Aβ17–40/42; ∼3 kDa). To determine whether similar processing events underlie βAPP metabolism in neurons, media were examined following conditioning by primary neuronal cultures derived from embryonic day 17 rats. Immunoprecipitates of conditioned media derived from [35S]methionine pulse‐labeled primary neuronal cultures contained 4‐ and 3‐kDa Aβ‐related species. Radiosequencing analysis revealed that the 4‐kDa band corresponded to conventional Aβ beginning at position Aβ(Asp1), whereas both radio‐sequencing and immunoprecipitation‐mass spectrometry analyses indicated that the 3‐kDa species in these conditioned media began with Aβ(Glu11) at the N terminus, rather than Aβ(Leu17) as does the conventional p3 peptide. Either activation of protein kinase C or inhibition of protein phosphatase 1/2A increased soluble βAPPα release and decreased generation of both the 4‐kDa Aβ and the 3‐kDa N‐truncated Aβ. Unlike results obtained with continuously cultured cells, protein phosphatase 1/2A inhibitors were more potent at reducing Aβ secretion by neurons than were protein kinase C activators. These data indicate that rodent neurons generate abundant Aβ variant peptides and emphasize the role of protein phosphatases in modulating neuronal Aβ generation.
Evidence for the Existence of Imidazoline‐Specific Binding Sites in Synaptosomal Plasma Membranes of the Bovine Brainstem
Authors: Heemskerk F. M. J. , Dontenwill M. , Greney H. , Vonthron C. , Bousquet P.
Published: July 2010

Abstract: Nonadrenergic imidazoline‐specific binding sites were characterized pharmacologically in crude cerebral membrane preparations, but little is known about their subcellular localization in neurons. As in the brain‐stem these sites are involved in cardiovascular regulation and peripherally imidazolines modulate neurotransmitter release, we tried to determine a possible (pre)synaptic localization in brainstem. We found a specific enrichment in (entire) synaptosome, purified synaptosomal plasma membrane (37 fmol/mg), and mitochondrial (83 fmol/mg) fractions as compared with other membrane fractions (3–8 fmol/mg). Synaptosomes appeared to be free of postsynaptic structures, and purified synaptosomal plasma membranes were devoid of mitochondrial material, as determined by electron microscopy and by comparison with the distribution of marker enzymes such as monoamine oxidase. These results show for the first time that these extramitochondrial imidazoline‐specific sites are neuronal and are located on presynaptic terminals. We found high affinities for unlabeled p‐iodoclonidine (subnanomolar), clonidine (0.2 nM), and efaroxan (11 nM), but idazoxan did not compete significantly for the p‐[125I]iodoclonidine binding in these membranes. Therefore, these sites can be classified as I1 imidazoline receptors. In summary, we describe for the first time that high‐affinity I1 receptors of the bovine brainstem are located on (pre)synaptic membranes.
Expression of a Functional Anaphylatoxin C3a Receptor by Astrocytes
Authors: Ischenko Alexander , Sayah Sakina , Patte Christine , Andreev Serguey , Gasque Philippe , Schouft Marie‐Thérèse , Vaudry Hubert , Fontaine Marc
Published: July 2010

Abstract: Human astrocyte cell lines reportedly contain a specific receptor for the complement anaphylatoxin C3a based on ligand‐binding studies, functional responses, and RNA analysis by RT‐PCR. Uptake of 125I‐C3a by astrocytes was specific and reversible. Scatchard analysis indicated the presence of two classes of binding sites. High‐affinity binding sites were abundantly expressed (20,000–80,000 sites per cell) with an estimated KD of 1–2 nM. Low‐affinity binding sites with a KD of 209 nM were largely expressed (n≥ 4 × 106 sites per cell) and probably did not reflect a receptor‐mediated binding, but rather an ionic interaction between C3a and the membrane. Analysis of astrocyte mRNA by RT‐PCR with three different sets of primers covering 60% of the C3a receptor (C3aR) mRNA sequence indicated that glial C3aR was identical to the leukocytic one. Western blot analysis using a specific anti‐C3aR evidenced a C3aR with a molecular mass of 60,000 Da. C3a and a superagonist peptide, E7, induced a transient increase of intracellular [Ca2+] in primary culture of astrocytes. Treatment of the ligands by carboxypeptidase B to eliminate the C‐terminus Arg considerably decreased the [Ca2+] response. Moreover, flow cytometry experiments demonstrated the expression of C3aR on normal rat astrocyte membrane. This report brings new insight for the role of the complement system in the brain inflammation response.
Protective Properties of Tin‐ and Manganese‐Centered Porphyrins Against Hydrogen Peroxide‐Mediated Injury in Rat Astroglial Cells
Authors: Dwyer Barney E. , Lu Shi‐Yi , Laitinen Jarmo T. , Nishimura Robert N.
Published: July 2010

Abstract: Tin‐mesoporphyrin (tin‐mp), a potent inhibitor of heme oxygenase, and manganese (III) tetrakis(1‐methyl‐4‐pyridyl)porphyrin (MnTMPyP), a potent superoxide dismutase mimetic, reduced H2O2 toxicity in cultures of transformed rat astroglial cells if added 30 min before, or at the same time as, H2O2. Reduced toxicity was not observed if treatment was delayed for 60 min, the time by which H2O2 was essentially eliminated from cultures. Coadministration of tin‐mp and MnTMPyP did not increase protection over either compound administered individually. Tin‐mp, but not MnTMPyP, was stable in culture. MnCl2 was not protective, suggesting that protection by MnTMPyP was not dependent on manganous ion, a by‐product of MnTMPyP breakdown. Protection by tin‐mp and MnTMPyP was not associated with metalloporphyrin‐mediated induction of heme oxygenase‐1 or with changes in heme oxygenase‐2 on western blots. Whereas protective concentrations of tin‐mp did not have superoxide dismutase‐mimetic properties in vitro, protective concentrations of MnTMPyP partially inhibited heme oxygenase. The data support the hypothesis that heme oxygenase inhibition is protective against acute oxidative injury.
Involvement of Caspase‐3‐Like Protease in the Mechanism of Cell Death Following Focally Evoked Limbic Seizures
Authors: Henshall David C. , Chen Jun , Simon Roger P.
Published: July 2010

The cysteine protease caspase‐3 may be involved in the mechanism of cell death following seizures. Using a rat model of focally evoked limbic epilepsy with continuous electroencephalography monitoring, we investigated seizure‐induced changes in caspase‐3 protein expression and processing, enzyme activity, and the in vivo effect of caspase‐3 inhibition. Seizures were induced by intraamygdaloid injection of kainic acid (0.1 μg) and were terminated after 45 min by diazepam (30 mg/kg) administration. Animals were killed 0‐72 h following diazepam administration. Levels of the 32‐kDa proenzyme form of caspase‐3 were unaffected by seizures. Levels of the 17‐kDa cleaved (active) fragment of caspase‐3 were almost undetectable in control brain, but were increased significantly at 4 and 24 h within ipsilateral hippocampus and cortex in seizure animals. Caspase‐3‐like protease activity was increased within the ipsilateral hippocampus at 8 and 24 h following seizures. Caspase‐3 immunoreactivity was increased within the vulnerable ipsilateral CA3/CA4 subfield at 24 and 72 h following seizures and was associated predominantly, but not exclusively, with neurons exhibiting DNA fragmentation. The putatively selective caspase‐3 inhibitor N‐benzyloxycarbonyl‐Asp(OMe)‐Glu(OMe)‐Val‐Asp(OMe)‐fluoromethyl ketone significantly improved neuronal survival bilaterally within the hippocampal CA3/CA4 subfields following seizures. Collectively, these data suggest that caspase‐3 may play a significant role in the mechanism by which neurons die following seizures.
Immunolocalization of the mGluR1b Splice Variant of the Metabotropic Glutamate Receptor 1 at Parallel Fiber‐Purkinje Cell Synapses in the Rat Cerebellar Cortex
Authors: Mateos José María , Benítez Rocío , Elezgarai Izaskun , Azkue Jon Jatsu , Lázaro Esther , Osorio Alexandra , Bilbao Aurora , Doñate Francisco , Sarría Rafael , Conquet François , Ferraguti Francesco , Kuhn Rainer , Knöpfel Thomas , Grandes Pedro
Published: July 2010

Several metabotropic glutamate receptor (mGluR) subtypes have been identified in the cerebellar cortex that are targeted to different compartments in cerebellar cells. In this study, preembedding immunocytochemical methods for electron microscopy were used to investigate the subcellular distribution of the mGluR1b splice variant in the rat cerebellar cortex. Dendritic spines of Purkinje cells receiving parallel fiber synaptic terminals were immunoreactive for mGluR1b. With a preembedding immunogold method, ~25% of the mGluR1b immunolabeling was observed perisynaptically within 60 nm from the edge of the postsynaptic densities. Values of extrasynaptic gold particles beyond the first 60 nm were maintained at between 10 and 18% along the whole intracellular surface of the dentritic spine membranes of Purkinje cells. For comparison, the distribution of mGluR1a was studied. A predominant (~37%) perisynaptic localization of mGluR1a was seen in dendritic spines of Purkinje cells, dropping the extrasynaptic labeling to 15% in the 60‐120‐nm bin from the edge of the postsynaptic specialization. Our results reveal that mGluR1b and mGluR1a are localized to the same subcellular compartments in Purkinje cells but that the densities of the perisynaptic and extrasynaptic pools were different for both isoforms. The compartmentalization of mGluR1b and mGluR1a might serve distinct requirements in cerebellar neurotransmission.
Probing the functions of NAAG via NAAG peptidases and MGLUR3
Authors: Wroblewska B. , Ramadan E. , Bacich D. J. , O'Keefe D. S. , Heston W. D. W. , Bukhari N. , Wegorzewska I. , Bzdega T. , Wroblewski J. T. , Kozikowski A. , Yamamoto T. , Neale J. H.
Published: July 2010

We have characterized N‐acetylaspartylglutamate (NAAG) and found that the peptide inhibits transmitter release by activation of mGluR3 receptors at presynaptic endings. To learn more about the synaptic activity of NAAG, we have synthesized a series of novel compounds that are potent inhibitors of brain NAAG peptidase activity as well as the activity of cloned human and rat glutamate carboxypeptidase II (GCPII), an enzyme that was believed to be solely responsible for inactivation of NAAG following synaptic release. We tested two of these compounds in models of chronic and neuropathic pain and found them to be antinociceptive. We have produced and characterized a strain of GCPII knock‐out mice and discovered a residual NAAG peptidase activity in their brains and spinal cords. They appear quite similar to their wild type littermates in terms of growth, reproduction, basic neurological features, acute pain threshold, rotor rod and open field behavior, and NAAG, glutamate, and mGluR3 mRNA levels. These data support the hypothesis that one or more uncharacterized enzymes may be involved in the inactivation of NAAG. We have found several differences between the NAAG peptidase activity found in the knock‐out vs. wild type brain membranes, including differential sensitivity to the peptidase inhibitor 2‐PMPA. The cloning of an additional gene(s) coding for nervous system NAAG peptidase and the development of selective inhibitors of NAAG peptidase activity have the potential to contribute to understanding the role of NAAG in excitotoxicity and chronic pain perception.
Axon‐to‐glia signalling is mediated by n‐acetylaspartylglutamate (NAAG) in crayfish
Authors: Lieberman E. M. , Urazaev A. K. , Fletcher P. L. , Gafurov B. S. , Grossfeld R. M.
Published: July 2010

Action potential generation in the medial giant axon of the crayfish causes the associated periaxonal glia to hyperpolarize by mechanisms sensitive to metabotropic glutamate receptor (mGLUR) and cholinergic receptor blockade. This response can be mimicked by the application of nanomolar NAAG or glutamate (GLU). Axons and glia synthesize NAAG from GLU or glutamine (GLN) and release it during electrical stimulation of the axon. NAAG is hydrolyzed to N‐acetylaspartate and GLU by glutamate carboxypeptidase II (GCPII), whose activity is increased by nerve fiber stimulation. When GCPII activity is blocked pharmacologically, NAAG's electrophysiological action on the glia is mediated solely by Group II mGLURs. When the enzyme is activated, GLU, the hydrolysis product of NAAG, contributes to glial hyperpolarization through a mechanism sensitive to NMDA receptor blockade. Both NAAG and GLU cause an increase in glial Ca2+ that may be a common link to a transduction cascade that hyperpolarizes glia by reducing Cl– permeability. A consequence of these changes is the modulation of K+ homeostasis of the neuronal microenvironment, preserving the ability of the axon to generate impulses at high frequency. GLU formed by NAAG hydrolysis is avidly accumulated by periaxonal glia, where it is converted to GLN and NAAG. The uptake of GLN and conversion to GLU and NAAG by axons provides a possible mechanism for recycling of the axon‐to‐glia signalling agent(s) in crayfish nerve fibers. Together, these results suggest that NAAG is the primary axon‐to‐glia signalling agent and source of signalling GLU at nonsynaptic regions of crayfish nerve fibers.

Acknowledgements:  Supported by NIH grant NS34799 and Guilford Pharmaceuticals (Baltimore, MD).
CIP98, a novel PDZ domain protein, is expressed in the central nervous system and interacts with calmodulin‐dependent serine kinase
Authors: Yap Chan Choo , Liang Fengyi , Yamazaki Yasuhiro , Muto Yuko , Kishida Haruo , Hayashida Tsuyako , Hashikawa Tsutomu , Yano Ryoji
Published: July 2010

Receptors and various molecules in neurons are localized at precise locations to perform their respective functions, especially in synaptic sites. Among synaptic molecules, PDZ domain proteins play major roles in scaffolding and anchoring membrane proteins for efficient synaptic transmission. In the present study, we isolated CIP98, a novel protein (98 kDa) consisting of three PDZ domains and a proline‐rich region, which is widely expressed in the central nervous system. In situ hybridization and immunohistochemical staining patterns demonstrate that CIP98 is expressed strongly in certain types of neurons, i.e. pyramidal cells in layers III–V of the cerebral cortex, projecting neurons in the thalamus and interneurons in the cerebellum. The results of immunocytochemical staining and electron microscopy revealed that CIP98 is localized both in dendrites and axons. Interestingly, CIP98 interacts with CASK (calmodulin‐dependent serine kinase), a member of the membrane‐associated guanylate kinase (MAGUK) family that plays important roles in the molecular organization of proteins at synapses. CIP98 was shown to co‐localize with CASK along the dendritic processes of neurons. In view of its direct association with CASK, CIP98 may be involved in the formation of CASK scaffolding proteins complex to facilitate synaptic transmission in the CNS.
Dephosphorylation of pCREB by protein serine/threonine phosphatases is involved in inactivation ofAanatgene transcription in rat pineal gland
Authors: Koch Marco , Mauhin Viviane , Stehle Jörg H. , Schomerus Christof , Korf Horst‐Werner
Published: July 2010

The rat pineal gland is a suitable model to investigate neurotransmitter‐controlled gene expression, because it is well established that the stimulation of melatonin biosynthesis by norepinephrine (NE) depends on the activation of the gene that encodes arylalkylamineN‐acetyltransferase (AANAT), the melatonin rhythm enzyme. The mechanisms responsible for downregulation ofAanattranscription are less clear. In thisin vitrostudy we investigated the role of pCREB dephosphorylation for termination ofAanatgene transcription. Immunosignals for pCREB, strongly induced after NE stimulation, rapidly decreased after withdrawal of NE. The immunoreactivity of the inhibitory transcription factor ICER increased twofold after NE treatment for 6 h, but did not change within 30 min after removal of the stimulus. Application of protein serine/threonine phosphatase (PSP) inhibitors prevented pCREB dephosphorylation and blocked the decreases inAanatmRNA levels, AANAT protein amount and melatonin biosynthesis all of which occurred rapidly after NE withdrawal. PSPs in the rat pineal gland were characterized by immunocytochemistry and immunoblotting. NE‐stimulation for 8 h induced accumulation of PSP1‐catalytic subunit (CSU) in pinealocyte nuclei, but did not affect the distribution of PSP2A‐CSU. The results identify dephosphorylation of pCREB by PSPs as an essential mechanism for downregulation ofAanattranscription in the rat pineal gland.
Astrocyte metabolism is disturbed in the early development of experimental hydrocephalus
Authors: Kondziella Daniel , Qu Hong , Lüdemann Wolf , Brinker Thomas , Sletvold Olav , Sonnewald Ursula
Published: July 2010

The proper diagnosis of the arrested or the progressive form of hydrocephalus has a critical impact on treatment, but remains difficult. The assessment of early changes in cerebral metabolism might help in the development of adequate non‐invasive diagnostic tools. This study examined the alterations in label incorporation in neurotransmitter amino acids and other compounds in kaolin‐induced progressive hydrocephalus in rats by means of magnetic resonance spectroscopy (MRS) combined with the administration of [1‐13C]glucose and [1,2‐13C]acetate. Some 2, 4 and 6 weeks after kaolin injection into the cisterna magna, cerebrum, brainstem and cerebellum were dissected. Interestingly, labelling of most amino acids derived from [1‐13C]glucose showed no alterations, whereas labelling from [1,2‐13C]acetate was affected. Two weeks after induction of hydrocephalus the taurine concentration was decreased, whereas the concentration of [1,2‐13C]lactate was increased in the cerebrum and that of [1,2‐13C]GABA in the brainstem. Furthermore, labelling from [1,2‐13C]acetate was significantly decreased in [4,5‐13C]glutamate, [1,2‐13C]glutamate and [1,2‐13C]GABA in cerebrum from 4 weeks after hydrocephalus induction. The concentration ofN‐acetylaspartate, a neuronal marker, was unchanged. However, labelling of the acetyl group from [1‐13C]glucose was decreased in cerebellum and brainstem at 6 weeks after the induction of hydrocephalus. As glucose is metabolized predominately by neurones, whereas acetate is exclusively taken up by astrocytes, these results indicate that mostly astrocytic, and only later neuronal, metabolism is disturbed in the kaolin model of hydrocephalus. If verified in patients usingin vivoMRS, impaired astrocyte metabolism might serve as an early indication for operative treatment.
Early events of target deprivation/axotomy‐induced neuronal apoptosis in vivo: oxidative stress, DNA damage, p53 phosphorylation and subcellular redistribution of death proteins
Authors: Martin Lee J. , Price Anne C. , McClendon Karen B. , Al‐Abdulla Nael A. , Subramaniam Jamuna R. , Wong Philip C. , Liu Zhiping
Published: July 2010

The mechanisms of injury‐ and disease‐associated apoptosis of neurons within the CNS are not understood. We used a model of cortical injury in rat and mouse to induce retrograde neuronal apoptosis in thalamus. In this animal model, unilateral ablation of the occipital cortex induces apoptosis of corticopetal projection neurons in the dorsal lateral geniculate nucleus (LGN), by 7 days post‐lesion, that is p53 modulated and Bax dependent. We tested the hypothesis that this degenerative process is initiated by oxidative stress and early formation of DNA damage and is accompanied by changes in the levels of pro‐apoptotic mediators of cell death. Immunoblotting revealed that the protein profiles of Bax, Bak and Bad were different during the progression of neuronal apoptosis in the LGN. Bax underwent a subcellular redistribution by 1 day post‐lesion, while Bak increased later. Bad showed an early sustained increase. Cleaved caspase‐3 was elevated maximally at 5 and 6 days. Active caspase‐3 underwent a subcellular translocation to the nucleus. A dramatic phosphorylation of p53 was detected at 4 days post‐lesion. DNA damage was assessed immunocytochemically as hydroxyl radical adducts (8‐hydroxy‐2‐deoxyguanosine) and single‐stranded DNA. Both forms of DNA damage accumulated early in target‐deprived LGN neurons. Transgenic overexpression of superoxide dismutase‐1 provided significant protection against the apoptosis but antioxidant pharmacotreatments with trolox and ascorbate were ineffective. We conclude that overlapping and sequential signaling pathways are involved in the apoptosis of adult brain neurons and that DNA damage generated by superoxide derivatives is an upstream mechanism for p53‐regulated, Bax‐dependent apoptosis of target‐deprived neurons.
Orphan nuclear receptor Nurr1 directly transactivates the promoter activity of the tyrosine hydroxylase gene in a cell‐specific manner
Authors: Kim Kwang‐Soo , Kim Chun‐Hyung , Hwang Dong‐Youn , Seo Hyemyung , Chung Sangmi , Hong Seok Jong , Lim Jin‐Kyu , Anderson Therese , Isacson Ole
Published: July 2010

Tyrosine hydroxylase (TH) catalyzes the first and rate‐limiting step of catecholamine synthesis and its expression is necessary for neurotransmitter specification of all catecholaminergic neurons, while dopamine β‐hydroxylase (DBH) is essential for the noradrenergic phenotype. In the present study, we show that Nurr1, an orphan nuclear receptor critical for dopaminergic (DA) neuron development, directly transactivates the promoter activity of the TH gene in a cell type‐dependent manner, while it does not regulate the DBH promoter. Consistent with these results, only the TH promoter contains multiple sequence motifs homologous to the known Nurr1‐binding motif, NBRE. TH promoter deletional analysis indicates that < 1.0 kb upstream sequences, encompassing three NBRE‐like motifs (i.e. NL1, NL2 and NL3) are mostly responsible for the effects of Nurr1. Among these potential motifs, site‐directed mutational analysis showed that NL1, residing from − 35 to − 28 bp, was most critical for mediating the transactivation by Nurr1. Strikingly, however, both DNase I footprinting and electrophoretic mobility shift assays showed that NL3, but not NL1 or NL2, has high binding affinity to Nurr1. To determine whether the proximity of these motifs may be important for transactivation by Nurr1 in the transient transfection assay, we generated reporter gene constructs in which NL3 is immediately proximal to the TATA box. Indeed, NL3 was more efficient in this position than NL1 or NL2 for mediating the transactivation by Nurr1. Our results suggest that Nurr1 may play a direct role for specification of DA neurotransmitter identity by activating TH gene transcription in a cell context‐dependent manner.
Ero1‐L, an ischemia‐inducible gene from rat brain with homology to global ischemia‐induced gene 11 (Giig11), is localized to neuronal dendrites by a dispersed identifier (ID) element‐dependent mechanism
Authors: Chen Dexi , Jin Kunlin , Kawaguchi Kenji , Nakayama Masaki , Zhou Xiaoman , Xiong Zhigang , Zhou An , Mao Xiao Ou , Greenberg David A. , Graham Steven H. , Simon Roger P.
Published: July 2010

Many changes in neuronal gene expression occur in response to ischemia, and these may play a role in determining the fate of ischemic neurons. To identify genes induced in the rat brain following cerebral ischemia, a strategy was used that combines subtractive hybridization and differential screening. Among the genes identified was one referred to as global ischemia‐inducible gene 11(Giig11). Sequence analysis indicated that Giig11 exhibited 97% and 91% identity to the known Ero1‐L (S. cereviseae ero1‐like oxidoreductase) of mouse and human origin, which is involved in oxidative endoplasmic reticulum protein folding. Rat Ero1‐L/Giig11 also contains a l07‐bp sequence that is nearly identical (> 95%) to the known dispersed repetitive identifier (ID), but which is lacking in mouse and human Ero1‐L. Northern blotting showed that expression of the ID element and Ero1‐L/Giig11 mRNA increased after global cerebral ischemia. In situ hybridization demonstrated increased expression of Ero1‐L/Giig11 in the brain following ischemic injury, with the highest levels in the vulnerable hippocampal CA1 pyramidal neurons. Transfection of cultured primary hippocampal neurons with a plasmid containing green fluorescent protein (gfp) and Ero1‐L/Giig11 cDNA (with and without the ID element) produced a gfp‐Ero1‐L/Giig11 fusion protein, and more fusion protein was localized into dendrites in the presence of the ID element, suggesting that the ID element promotes Ero1‐L/Giig11 protein localization to dendrites. Therefore, Ero‐1L/Giig11 may have a role in ischemia‐induced neuronal repair or survival mechanisms directed at counteracting abnormalities in protein folding, maturation and distribution.
Adenosine A2Areceptor‐mediated facilitation of noradrenaline release involves protein kinase C activation and attenuation of presynaptic inhibitory receptor‐mediated effects in the rat vas deferens
Authors: Queiroz Glória , Talaia Carlos , Gonçalves Jorge
Published: July 2010

In the epididymal portion of rat vas deferens, facilitation of noradrenaline release mediated by adenosine A2Areceptors, but not that mediated by β2‐adrenoceptors or by direct activation of adenylyl cyclase, was attenuated by blockade of α2‐adrenoceptors and abolished by simultaneous blockade of α2‐adrenoceptors, adenosine A1and P2Y receptors. The adenosine A2Areceptor‐mediated facilitation was not changed by inhibitors of protein kinase A, protein kinase G or calmodulin kinase II but was prevented by inhibition of protein kinase C with chelerythrine or bisindolylmaleimide XI. Activation of protein kinase C with phorbol 12‐myristate 13‐acetate caused a facilitation of noradrenaline release that was abolished by bisindolylmaleimide XI and reduced by antagonists of α2‐adrenoceptors, adenosine A1and P2Y receptors. Activation of adenosine A2Areceptors attenuated the inhibition of noradrenaline release mediated by the presynaptic inhibitory receptors. This effect was mimicked by phorbol 12‐myristate 13‐acetate and prevented by bisindolylmaleimide XI. It is concluded that adenosine A2Areceptors facilitate noradrenaline release by a mechanism that involves a protein kinase C‐mediated attenuation of effects mediated by presynaptic inhibitory receptors, namely α2‐adrenoceptors, adenosine A1and P2Y receptors.