bluuebunny/crossref_metadata_2025 huggingface dataset:
Putrescine‐Modified Nerve Growth Factor: Bioactivity, Plasma Pharmacokinetics, Blood‐Brain/Nerve Barrier Permeability, and Nervous System Biodistribution
Authors: Poduslo Joseph F. , Curran Geoffry L. , Gill Jagjit S.
Published: July 2010

Abstract: Previous investigations from our laboratory have demonstrated that the covalent modification of a variety of proteins, including antioxidant enzymes, with the naturally occurring polyamines—putrescine (PUT), spermidine, and spermine—dramatically increases their permeability coefficient‐surface area product (PS) at the blood‐brain and blood‐nerve barriers after parenteral administration. In the present study, we have covalently modified nerve growth factor (NGF) with PUT by targeting carboxylic groups for their graded modification by controlling the ionization of these groups with pH. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, western, and isoelectric focusing analyses demonstrated conversion of NGF to its polyamine‐modified derivatives at different pH values. Although the immunoreactivity of PUT‐NGF determined by ELISA and western analysis decreased with decreasing pH, the biological activity of PUT‐NGF was not affected at any pH as determined by survival and neurite extension of dorsal root ganglia and PC12 cultures. Plasma pharmacokinetics after a single intravenous bolus administration revealed intact PUT‐NGF through 10 min and 73–82% intact protein at 15 min. The PS value for PUT‐NGF was maximized and the residual plasma volume (Vp) of the protein in the blood vessels minimized when the pH of the modification reaction was >6.4. The biodistribution of PUT‐NGF at 15 min showed 22–33% intact protein in different brain regions, which represented 0.4–5.9 ng of PUT‐NGF in different brain regions, a physiological dose that is capable of eliciting a bioresponse. The design of this polyamine‐modified NGF derivative that has enhanced permeability at the blood‐brain and blood‐nerve barriers with retained bioactivity may obviate the necessity to create small‐molecule mimics of NGF and may be applicable to neurotrophins, engineered multifunctional chimeric neurotrophins, antioxidant enzymes, and other therapeutic proteins with specific clinical application to neurological diseases.
Chronic Imipramine Administration Amplifies the Serotonin2A Receptor‐Induced Intracellular Ca2+ Mobilization in C6 Glioma Cells Through a Calmodulin‐Dependent Pathway
Authors: Muraoka Shinichiro , Kamei Kenji , Muneoka Katsumasa , Takigawa Morikuni
Published: July 2010

Abstract: In the present study, we examined whether chronic exposure of C6BU‐1 cells to 100 nM of several different types of antidepressants directly influences serotonin2A (5‐HT2A) receptor‐stimulated intracellular Ca2+ mobilization. Imipramine, desipramine, clomipramine, and maprotiline amplified the 5‐HT response at 48, but not at 2, h. Imipramine increased the maximum response to 5‐HT without altering the EC50 of the dose‐response curve. This effect was time dependent and cycloheximide blocked the maximal induction, suggesting an essential role for protein synthesis in this process. Previous exposure of the cells to thrombin or isoproterenol did not influence 5‐HT2A receptor function and pretreatment with imipramine did not alter the thrombin‐ or bradykinin‐induced Ca2+ mobilization, which indicates that the effects of imipramine appear to be specific to the 5‐HT2A receptor. The effect of imipramine was potently suppressed by a calmodulin antagonist, W‐13, in a dose‐dependent manner. Furthermore, this amplified 5‐HT response was blocked by KN‐93, but not by H‐7. Taken together, these results suggest that imipramine has a modulatory effect on the 5‐HT2A receptor‐coupled intracellular Ca2+ in C6 cells through a calmodulin‐dependent pathway, possibly involving Ca2+/calmodulin kinase activation.
Regulation of Oleoyl‐CoA Synthesis in the Peripheral Nervous System: Demonstration of a Link with Myelin Synthesis
Authors: Garbay Bertrand , Boiron‐Sargueil Françoise , Shy Michael , Chbihi Taibi , Jiang Huiyuan , Kamholz John , Cassagne Claude
Published: July 2010

Abstract: We studied the regulation of oleic acid synthesis in the PNS. During mouse postnatal development, the proportion of 18:1 rises in the sciatic nerve from 17% at 5 days of age to 33% at 25 days. However, this rise does not occur in the dysmyelinating mutant mouse trembler. In normal mouse development, the total stearoyl‐CoA desaturase (SCD) activity measured in sciatic nerve homogenates is high during the first 3 weeks. Yet in trembler nerves, this SCD activity represents only 15% of normal values. Using the RT‐PCR technique, we demonstrate that the SCD2 isoform is predominantly expressed in the PNS. Northern blot analysis showed that the mRNA levels for SCD2 parallel those of other specific myelin proteins in both normal mouse and trembler mutant development. Similar experiments in a rat demyelination‐remyelination model confirmed that SCD2 mRNA levels are regulated in the PNS in a similar manner to myelin‐specific proteins.
In Vitro Evaluation of 11C‐Labeled (S)‐Nicotine, (S)‐3‐Methyl‐5‐(1‐Methyl‐2‐Pyrrolidinyl)isoxazole, and (R,S)‐1‐Methyl‐2‐(3‐Pyridyl)azetidine as Nicotinic Receptor Ligands for Positron Emission Tomography Studies
Authors: Sihver Wiebke , Fasth Karl‐Johan , Ögren Mattias , Bivehed Håkan , Bergström Mats , Nordberg Agneta , Watanabe Yasuyoshi , Långström Bengt
Published: July 2010

Abstract: The binding characteristics of the novel 11C‐labeled nicotinic ligands (R,S)‐1‐methyl‐2‐(3‐pyridyl) azetidine (MPA) and (S)‐3‐methyl‐5‐(1‐methyl‐2‐pyrrolidinyl)isoxazole (ABT‐418) were investigated in comparison with those of (S)‐[11C]nicotine in vitro in the rat brain to be able to predict the binding properties of the new ligands for positron emission tomography studies in vivo. The data from time‐resolved experiments for all ligands indicated fast binding kinetics, with the exception of a slower dissociation of [11C]MPA in comparison with (S)‐[11C]nicotine and [11C]ABT‐418. Saturation experiments revealed for all ligands two nicotinic receptor binding sites with affinity constants (KD values) of 2.4 and 560 nM and binding site densities (Bmax values) of 65.5 and 223 fmol/mg of protein for (S)‐[11C]nicotine, KD values of 0.011 and 2.2 nM and Bmax values of 4.4 and 70.7 fmol/mg of protein for [11C]MPA, and KD values of 1.3 and 33.4 nM and Bmax values of 8.8 and 69.2 fmol/mg of protein for [11C]ABT‐418. In competing with the 11C‐ligands, epibatidine was most potent, followed by cytisine. A different rank order of potencies was found for (−)‐nicotine, (+)‐nicotine, MPA, and ABT‐418 displacing each of the 11C‐ligands. Autoradiograms displayed a similar pattern of receptor binding for all ligands, whereby [11C]MPA showed the most distinct binding pattern and the lowest nonspecific binding. We conclude that the three 11C‐labeled nicotinic ligands were suitable for characterizing nicotinic receptors in vitro. The very high affinity of [11C]MPA to nicotinic acetylcholine receptors, its low nonspecific binding, and especially the slower dissociation kinetics of the [11C]MPA from the putative high‐affinity nicotinic acetylcholine receptor binding site compared with (S)‐[11C]nicotine and [11C]ABT‐418 raise the level of interest in [11C]MPA for application in positron emission tomography.
A Novel Juxtamembrane Deletion in Rat TrkA Blocks Differentiative but Not Mitogenic Cell Signaling in Response to Nerve Growth Factor
Authors: Meakin Susan O. , MacDonald I. S.
Published: July 2010

Abstract: We have generated a novel rat TrkA receptor mutant (TrkAS3) by deletion of five conserved residues (493IMENP497) in the juxtamembrane domain. TrkAS3 receptors cannot support nerve growth factor (NGF)‐induced cell cycle arrest or neuronal differentiation but retain cell survival responses as well as Ras‐dependent mitogenic signaling. Cells of the nnr5 line stably expressing TrkAS3 induce NGF‐dependent SHC phosphorylation and phosphatidylinositol 3‐kinase, phospholipase Cγ‐1, and prolonged mitogen‐activated protein kinase activation to absolute levels comparable to those in PC12 cells. Although the stoichiometry of TrkAS3‐SHC binding is reduced, cells overexpressing TrkAS3 exhibit NGF‐dependent SHC‐Grb‐2/Sos binding, essential for Ras activation, as well as NGF‐dependent SNT phosphorylation to absolute levels comparable to those in PC12 cells. Collectively, these data suggest that the TrkAS3 deletion either directly affects a novel Ras‐independent TrkA binding protein or that the decrease in TrkAS3‐SHC association affects a Ras‐independent SHC binding protein essential for cell cycle arrest and/or neurite outgrowth.
Increased Activity of Eukaryotic Initiation Factor 2B in PC12 Cells in Response to Differentiation by Nerve Growth Factor
Authors: Muñoz F. , Quevedo C. , Martín M. E. , Alcázar A. , Salinas M. , Fando J. L.
Published: July 2010

Abstract: Translational rates, and activities and levels of initiation factors 2 and 2B were assessed in rat pheochromocytoma cells upon nerve growth factor (NGF) treatment. Two or 5 days of exposure to NGF caused significant quantitative increases in protein synthesis rate that are deemed necessary for neuronal differentiation. Changes in initiation factor 2 activity, as measured by its capacity to form a ternary complex, occur parallel to the observed changes in protein synthesis. Nevertheless, neither the intracellular levels of the initiation factor 2 nor the degree of phosphorylation of its α subunit can justify this increased activity. Interestingly, initiation factor 2B activity increases parallel to the neurite outgrowth, being significantly higher after 5 days of exposure to NGF, and could be responsible for the elevated rate of protein synthesis. No significant changes in the levels of eukaryotic initiation factor 2B, as determined with two different antibodies against the γ and ε subunits of the factor, were observed, implying that the increased activity should be regulated by factors other than its cellular concentration. Our results support the hypothesis that initiation factor 2B may play a role in the biochemical events controlling the differentiative growth factor‐induced signaling pathway in these cells.
Developmental Regulation of Tyrosine Phosphorylation of the NR2D NMDA Glutamate Receptor Subunit in Rat Central Nervous System
Authors: Dunah Anthone W. , Yasuda Robert P. , Wolfe Barry B.
Published: July 2010

Abstract: A subunit‐specific antibody against the N‐methyl‐d‐aspartate (NMDA) receptor NR2D protein along with an antiphosphotyrosine antibody were employed to examine the developmental profile of the tyrosine phosphorylation of NR2D and its regulation by a protein phosphatase inhibitor in rat brain. NMDA receptor proteins from the thalamus at postnatal days 1, 7, 21, and 49 were solubilized under denaturing conditions and used in immunoprecipitations with these antibodies followed by quantitative immunoblot analysis of NR2D protein in the resulting immunopellets. The results indicate that the NR2D subunit is tyrosine phosphorylated in the brain. The quantified data examining the developmental profile of tyrosine phosphorylation of NR2D in the thalamus show that the level of tyrosine phosphorylation of NR2D protein increases five‐ to sixfold during development. In addition, the protein phosphatase inhibitor pervanadate (vanadyl hydroperoxide) was found to increase tyrosine phosphorylation of NR2D subunit threefold in brain slices, implying an active cycle of phosphorylation and dephosphorylation in situ. These studies demonstrate developmentally regulated tyrosine phosphorylation of NR2D protein in vivo, suggesting that tyrosine phosphorylation may be important for regulating the functions of this NMDA receptor subunit in the mammalian central nervous system.
Protein Oxidative Damage in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis
Authors: Andrus Paula K. , Fleck Timothy J. , Gurney Mark E. , Hall Edward D.
Published: July 2010

Abstract: The Gly93→Ala mutation in the Cu,Zn superoxide dismutase (Cu,Zn‐SOD) gene (SOD1) found in some familial amyotrophic lateral sclerosis (FALS) patients has been shown to result in an aberrant increase in hydroxyl radical production by the mutant enzyme that may cause oxidative injury to spinal motor neurons. In the present study, we analyzed the extent of oxidative injury to lumbar and cervical spinal cord proteins in transgenic FALS mice that overexpress the SOD1 mutation [TgN(SOD1‐G93A)G1H] in comparison with nontransgenic mice. Total protein oxidation was examined by spectrophotometric measurement of tissue protein carbonyl content by the dinitrophenylhydrazine (DNPH) assay. Four ages were investigated: 30 (pre‐motor neuron pathology and clinical disease), 60 (after initiation of pathology, but pre‐disease), 100 (∼50% loss of motor neurons and function), and 120 (near complete hindlimb paralysis) days. Protein carbonyl content in 30‐day‐old TgN(SOD1‐G93A)G1H mice was twice as high as the level found in age‐matched nontransgenic mice. However, at 60 and 100 days of age, the levels were the same. Then, between 100 and 120 days of age, the levels in the TgN(SOD1‐G93A)G1H mice increased dramatically (557%) compared with either the nontransgenic mice or transgenic animals that overexpress the wild‐type human Cu,Zn‐SOD [TgN(SOD1)N29]. The 100–120‐day increase in spinal cord protein carbonyl levels was confirmed by sodium dodecyl sulfate‐polyacrylamide gel electrophoretic separation and western blot immunoassay, which enabled the identification of heavily oxidized individual proteins using a monoclonal antibody against DNPH‐derivatized proteins. One of the more heavily oxidized protein bands (14 kDa) was identified by immunoprecipitation as largely Cu,Zn‐SOD. Western blot comparison of the extent of Cu,Zn‐SOD protein carbonylation revealed that the level in spinal cord samples from 120‐day‐old TgN(SOD1‐G93A)G1H mice was significantly higher than that found in age‐matched nontransgenic or TgN(SOD1)N29 mice. These results suggest that the increased hydroxyl radical production associated with the G93A SOD1 mutation and/or lipid peroxidation‐derived radical species (peroxyl or alkoxyl) causes extensive protein oxidative injury and that the Cu,Zn‐SOD itself is a key target, which may compromise its antioxidant function.
Mutational Analysis of Substrate Inhibition in Tyrosine Hydroxylase
Authors: Quinsey Noelene S. , Luong Anh Q. , Dickson Phillip W.
Published: July 2010

Abstract: Substrate inhibition in tyrosine hydroxylase (TH) was analyzed by deletion mutagenesis. The deletion mutant TH 156/456 was the smallest section of TH to retain substrate inhibition. The TH 156/456 was monomeric, and so multimer formation does not play a role in substrate inhibition in TH. Further deletion at the N terminus to residue 169 produced a TH molecule with no substrate inhibition but high activity. A mutagenic scan of this region showed that mutations at Trp166 were responsible for this phenotype. A screen of a library of TH molecules containing random mutations identified three other mutants that had lost substrate inhibition but retained high activity. The results in this report are consistent with a model in which substrate inhibition acts through an allosteric mechanism.
Arginine Availability Controls the N‐Methyl‐d‐Aspartate‐Induced Nitric Oxide Synthesis: Involvement of a Glial‐Neuronal Arginine Transfer
Authors: Grima Gilbert , Cuénod Michel , Pfeiffer Silvia , Mayer Bernd , Do Kim Q.
Published: July 2010

Abstract: The neuronal nitric oxide (NO) synthase generates NO from arginine. NO mediates its physiological effects mainly by stimulating the synthesis of cyclic GMP. We have investigated the role of the arginine availability on the NMDA‐induced cyclic GMP accumulation in immature rat brain slices. The effect of NMDA was blocked by the inhibitor of the NO synthase, NG‐nitro‐l‐arginine, and by the antagonist of ionotropic non‐NMDA receptors, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX). This inhibition was not due to a direct interaction of CNQX with the NMDA receptor, and it was overcome by the presence of exogenously applied arginine. CNQX also blocked the NMDA‐evoked release of [3H]arginine from cerebellar slices. Moreover, the arginine uptake inhibitor l‐lysine reduced the cyclic GMP response to NMDA significantly. Therefore, the extracellular arginine availability, which is dependent on the activation of ionotropic non‐NMDA receptors, determines the rate of the NO biosynthesis by the neuronal NO synthase. Together with the reported release of arginine from glial cells upon activation of glial ionotropic non‐NMDA receptors and the predominant glial localization of arginine, these data provide the first evidence of an essential role of the arginine transfer from glial cells to neurons for the biosynthesis of NO.
Differential Properties of Two Stably Expressed Brain‐Specific Glycine Transporters
Authors: López‐Corcuera Beatriz , Martínez‐Maza Rodrigo , Núñez Enrique , Roux Michel , Supplisson Stéphane , Aragón Carmen
Published: July 2010

Abstract: Clonal cell lines stably expressing the glial glycine transporter 1b (GLYT1b) and the neuronal glycine transporter 2 (GLYT2) from rat brain have been generated and used comparatively to examine their kinetics, ion dependence, and electrical properties. Differential sensitivity of the transporters to sarcosine is clearly exhibited by the clonal cell lines. GLYT2 transports glycine with higher apparent affinity than GLYT1b and is not inhibited by any assayed compound, as deduced by glycine transport assays and electrophysiological recordings. A sigmoidal Na+ dependence of the glycine uptake by the stable cell lines is observed, indicating the involvement of more than one Na+ in the transport process. A more cooperative behavior for Na+ of GLYT2 than GLYT1b is suggested. One Cl− is required for GLYT1b and GLYT2 transport cycles, although GLYT1b shows three times higher affinity for this ion than GLYT2. The number of expressed transporters was sufficient to allow electrophysiological recordings of the uptake current in the two stable cell lines. GLYT2 exhibits more voltage dependence in both its glycine‐evoked current and its capacitive currents recorded in the absence of substrate.
The Mouse GalR2 Galanin Receptor: Genomic Organization, cDNA Cloning, and Functional Characterization
Authors: Pang Ling , Hashemi Tanaz , Lee Hu‐Jung J. , Maguire Maureen , Graziano Michael P. , Bayne Marvin , Hawes Brian , Wong Gwendolyn , Wang Suke
Published: July 2010

Abstract: The diverse physiological actions of galanin are thought to be mediated through activation of galanin receptors (GalRs). We report the genomic and cDNA cloning of a mouse GalR that possesses a genomic structure distinct from that of GalR1 and encodes a functional galanin receptor. The mouse GalR gene consists of two exons separated by a single intron within the protein‐coding region. The splicing site for the intron is located at the junction between the third transmembrane domain and the second intracellular loop. The cDNA encodes a 370‐amino acid putative G protein‐coupled receptor that is markedly different from human GalR1 and rat GalR3 (38 and 57%) but shares high homology with rat GalR2 (94%). In binding studies utilizing membranes from COS‐7 cells transfected with mouse GalR2 cDNA, the receptor displayed high affinity (KD = 0.47 nM) and saturable binding with 125I‐galanin (Bmax = 670 fmol/mg). The radioligand binding can be displaced by galanin and its analogues in a rank order: galanin ⋍ M40 ⋍ M15 ⋍ M35 ⋍ C7 ⋍ galanin (2–29) ⋍ galanin (1–16) ≫ galanin (10–29) ⋍ galanin (3–29), which resembles the pharmacological profile of the rat GalR2. Receptor activation by galanin in COS‐7 cells stimulated phosphoinositide metabolism, which was not reversed by pertussis toxin. Thus, the galanin receptor encoded in the cloned mouse GalR gene is the type 2 galanin receptor and is active in both ligand binding and signaling assays.
Molecular Identification and Coexpression of Galanin and GalR‐1 Galanin Receptor in the Human Ocular Ciliary Epithelium: Differential Modulation of Their Expression by the Activation of α2‐ and β2‐Adrenergic Receptors in Cultured Ciliary Epithelial Cells
Authors: Ortego Javier , Coca‐Prados Miguel
Published: July 2010

Abstract: Here we report the coexpression of the neuropeptide galanin and GalR‐1 galanin receptors in the human ciliary epithelium, a bilayer of neuroepithelial cells [nonpigmented (NPE) and pigmented (PE)] with neuroendocrine functions, and in a cell line (ODM‐2) derived from the NPE cells. Stimulation of ODM‐2 cells with phorbol ester [phorbol 12‐myristate 13‐acetate (PMA)] or forskolin resulted in an up‐regulation (two‐ to threefold) of galanin mRNA expression. Procaterol, a selective β2‐adrenergic agonist, and the catecholamine isoproterenol exerted a long‐term down‐regulation on galanin mRNA expression when added alone or in combination with PMA or forskolin. These actions exerted by procaterol or isoproterenol were abolished in the presence of ICI 118,551, a selective β2‐adrenergic antagonist. A radioimmunoassay for galanin peptide indicated that galanin or a galanin‐like product is present in the human aqueous humor fluid and is accumulated with time in the culture medium of ODM‐2 cells. It is interesting that norepinephrine, which exhibited no effect on galanin mRNA expression, induced a down‐regulation in the level of galanin or galanin‐like product accumulated in the medium of cultured ODM‐2 cells to levels even lower than those induced by β2‐adrenergic agonists. This effect is best explained by the concomitant up‐regulation (four‐ to fivefold) of GalR‐1 galanin receptor transcripts induced through the activation of α2‐adrenergic receptors. These findings support the view that pathways elicited by the activation of α2‐ and β2‐adrenergic receptors influence the expression of galanin and GalR‐1 galanin receptors in ciliary epithelial cells.
Colocalization and Complex Formation Between Prosaposin and Monosialoganglioside GM3 in Neural Cells
Authors: Misasi R. , Sorice M. , Garofalo T. , Griggi T. , Campana W. M. , Giammatteo M. , Pavan A. , Hiraiwa M. , Pontieri G. M. , O'Brien J. S.
Published: July 2010

Abstract: Prosaposin, the precursor of saposins A, B, C, and D, was recently identified as a neurotrophic factor in vitro as well as in vivo. Its neurotrophic activity has been localized to a linear 12‐amino acid sequence located in the NH2‐terminal portion of the saposin C domain. In this study, we show the colocalization of prosaposin and ganglioside GM3 on NS20Y cell plasma membrane by scanning confocal microscopy. Also, TLC and western blot analyses showed that GM3 was specifically associated with prosaposin in immunoprecipitates; this binding was Ca2+‐independent and not disassociated during sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The association of prosaposin‐GM3 complexes on the cell surface appeared to be functionally important, as determined by differentiation assays. Neurite sprouting, induced by GM3, was inhibited by antibodies raised against a 22‐mer peptide, prosaptide 769, containing the neurotrophic sequence of prosaposin. In addition, pertussis toxin inhibited prosaptide‐induced neurite outgrowth, as well as prosaptide‐enhanced ganglioside concentrations in NS20Y cells, suggesting that prosaposin acted via a G protein‐mediated pathway, affecting both ganglioside content and neuronal differentiation. Our findings revealed a direct and right GM3‐prosaposin association on NS20Y plasma membranes. We suggest that ganglioside‐protein complexes are structural components of the prosaposin receptor involved in cell differentiation.
Steroid Hormones Block Amyloid Fibril‐Induced 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) Formazan Exocytosis: Relationship to Neurotoxicity
Authors: Liu Yuanbin , Schubert David
Published: July 2010

Abstract: Perhaps the most reproducible early event induced by the interaction of amyloid β peptide (Aβ) with the cell is the inhibition of cellular 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction. We recently demonstrated that cytotoxic amyloid peptides such as Aβ and human amylin inhibit cellular MTT reduction by dramatically enhancing MTT formazan exocytosis. We now show the following: (a) Insulin and glucagon, when converted to fibrils with β‐pleated sheet structure, induce MTT formazan exocytosis that is indistinguishable from that induced by Aβ. NAC35, an amyloidogenic fragment of α‐synuclein (or NACP), also induces MTT formazan exocytosis. (b) All protein fibrils with the β‐pleated sheet structure examined are toxic to rat hippocampal neurons. (c) Many sterol sex hormones (e.g., estradiol and progesterone) block amyloid fibril‐enhanced MTT formazan exocytosis as well as MTT formazan exocytosis in control cells by acting at a common late step in the exocytic pathway. Steroids fail, however, to protect hippocampal neurons from acute amyloid fibril toxicity. These findings suggest that the ability to enhance MTT formazan exocytosis and to induce neurotoxicity are common biological activities of protein fibrils with β‐pleated sheet structure but that enhanced MTT formazan exocytosis is not sufficient for acute Aβ neurotoxicity.
Structural Aspects of Congo Red as an Inhibitor of Protease‐Resistant Prion Protein Formation
Authors: Demaimay Rémi , Harper James , Gordon Heather , Weaver Donald , Chesebro Bruce , Caughey Byron
Published: July 2010

Abstract: Congo red (CR) has been shown to inhibit the accumulation in scrapie‐infected cells of prion protein (PrP) in the abnormal protease‐resistant form (PrP‐res). However, it was not clear if this effect was due to a direct interaction of CR with either PrP‐res or its protease‐sensitive precursor (PrP‐sen) or to a less direct effect on living cells. Here we show that CR inhibits PrP‐res formation in a simple cell‐free reaction composed predominantly of purified PrP‐res and PrP‐sen. Structurally modified CR analogues were also compared in both the cell‐free conversion reaction and scrapie‐infected neuroblastoma cells. Methylation of the central phenyl groups at the 2,2′ positions diminished the inhibitory potency by ≥10‐fold. In contrast, there was little effect of 3,3′ methylation of the phenyls, deletion of one phenyl, or addition of an amido group between the phenyls. The relative activities of these compounds were well correlated in both cellular and acellular systems. Molecular modeling indicated that CR and 3,3′‐methyl‐CR have little rotational restriction about the biphenyl bond and can readily adopt a planar conformation, as can phenyl‐CR and amido‐CR. In contrast, 2,2′‐methyl‐CR is restricted to a nonplanar conformation of the biphenyl group. Thus, planarity and/or torsional mobility of the central phenyl rings of CR and its analogues is probably important for inhibition of PrP‐res formation. On the other hand, variations in the intersulfonate distance in these molecules had little effect on PrP‐res inhibition. These results indicated a high degree of structural specificity in the inhibition of PrP‐res formation by CR and related compounds.
Glutamine Uptake at the Blood‐Brain Barrier Is Mediated by N‐System Transport
Authors: Ennis Steven R. , Kawai Nobuyuki , Ren Xiao‐dan , Abdelkarim Galaleldin E. , Keep Richard F.
Published: July 2010

Abstract: The mechanism of unidirectional transport of glutamine from blood to brain in pentobarbital‐anesthetized rats was examined using in situ perfusion. Amino acid uptake into brain across the blood‐brain barrier (BBB) is classically thought to be via the Na‐independent large neutral (L‐system), acidic and basic amino acid transporters. In the presence of physiological concentrations of amino acids in the perfusate, which should saturate the known amino acid transporters at the BBB, the cortical transfer constant (Ki) for l‐[14C]glutamine was 11.6 ± 1.1 µl/g/min. The addition of either 10 mM 2‐amino‐2‐norbornanecarboxylic acid or 10 mM 2‐amino‐2‐norbornanecarboxylic acid and 5 mM cysteine had no effect on the cortical Ki for l‐[14C]glutamine, indicating that glutamine transport under these conditions does not occur by the L‐, A‐, or ASC‐systems. Decreasing perfusate Na from 140 to 2.4 mM by Tris substitution reduced the cortical Ki for l‐[14C]glutamine by 62% (p≤ 0.001). The Na‐dependent uptake has the characteristics of N‐system transport. It was inhibited by l‐histidine and l‐glutamine, both N‐system substrates, and it was pH sensitive and moderately tolerant of Li substitution for Na. This putative N‐system transporter at the luminal membrane of the BBB plays an important role in mediating brain glutamine uptake.
Ca2+/Calmodulin‐Mediated Regulation of Agonist‐Induced Sequestration of Gq Protein‐Coupled Histamine H1 Receptors in Human U373 MG Astrocytoma Cells
Authors: Hishinuma Shigeru , Naiki Akie , Tsuga Hirofumi , Young J. Michael
Published: July 2010

Abstract: We investigated the regulation by intracellular Ca2+ of agonist‐induced sequestration of Gq protein‐coupled histamine H1 receptors in human U373 MG astrocytoma cells. Histamine‐induced sequestration of H1 receptors from the cell surface membrane was detected as the loss of [3H]mepyramine binding sites on intact cells accessible to the hydrophilic H1‐receptor antagonist pirdonium. The changes in the pirdonium‐sensitive binding of [3H]mepyramine were mirrored by changes in the subcellular distribution of H1 receptors detected by sucrose density gradient centrifugation. The histamine‐induced sequestration of H1 receptors did not occur in hypertonic medium, in which clathrin‐mediated endocytosis is known to be inhibited, but was significantly accelerated in the absence of extracellular Ca2+ or in the presence of the calmodulin antagonists W‐7 and calmidazolium. Inhibitors of protein kinase C (H‐7 and GF109203X), Ca2+/calmodulin‐dependent protein kinase II (KN‐62), or protein phosphatase 2B (FK506) did not alter the time course of H1‐receptor sequestration. These results provide the first evidence that agonist‐induced, clathrin‐mediated sequestration of Gq protein‐coupled receptors is transiently inhibited by Ca2+/calmodulin, with the result that receptors remain on the cell surface membrane during the early stage of agonist stimulation.
Quantitative and Statistical Analysis of the Shape of Amperometric Spikes Recorded from Two Populations of Cells
Authors: Colliver Thomas L. , Hess Ellen J. , Pothos Emmanuel N. , Sulzer David , Ewing Andrew G.
Published: July 2010

Previously used methods of comparing amperometric spike characteristics from two separate groups of cells have entailed pooling all the values for a spike characteristic from each group of cells and then statistically comparing the two samples. Although this approach has indicated that there are significant differences between the spike characteristics from coloboma and control mouse chromaffin cells, the results are not consistent between experiments. We have reexamined the assumptions of the statistical tests used as well as the variability inherent in amperometric data measured from two groups of cells. Our findings indicate that when comparing amperometric spike characteristics between groups of cells, it is more appropriate to compare samples of mean spike values. This method consistently indicates that there is no difference between coloboma and control amperometric spikes. These results have been validated by using samples of mean spike characteristics to detect changes in the shape of amperometric spikes from both mouse chromaffin cells at 37°C and PC12 cells previously exposed to 50 μM L‐3,4‐dihydroxyphenylalanine and by the use of an additional analysis method, the nested ANOVA. Together, these results indicate that pooled samples of amperometric spike characteristics can give results that may confound the interpretation of amperometric data.
Chronic Mitochondrial Inhibition Induces Selective Motoneuron Death In Vitro: A New Model for Amyotrophic Lateral Sclerosis
Authors: Kaal Evert C. A. , Vlug Angela S. , Versleijen Michelle W. J. , Kuilman Maja , Joosten Elbert A. J. , Bär P. R. Dop
Published: July 2010

Evidence is increasing that mitochondrial dysfunction is involved in amyotrophic lateral sclerosis, a neurodegenerative disease characterized by selective motoneuron death. To study the role of mitochondrial dysfunction in the pathways leading to motoneuron death, we developed an in vitro model of chronic motoneuron toxicity, based on malonate‐induced inhibition of complex II in the mitochondrial electron transport chain. Treatment with malonate resulted in a dose‐dependent decrease in cellular ATP levels. We observed that motoneurons were significantly more vulnerable to mitochondrial inhibition than control neurons in the dorsal horn. We could reproduce this dose‐dependent phenomenon with the complex IV inhibitor sodium azide. The free radical scavenger α‐phenyl‐N‐tert‐butylnitrone, the AMPA/kainate receptor blocker 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, and riluzole, a drug that is currently used for the treatment of amyotrophic lateral sclerosis, were protective against malonate‐induced motoneuron death. Furthermore, the caspase inhibitors N‐benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethyl ketone and z‐Asp‐Glu‐Val‐Asp‐fluoromethyl ketone were both protective against malonate toxicity. Our model shows that chronic mitochondrial inhibition leads to selective motoneuron death, which is most likely apoptotic.
Neuroprotective Mechanism of Glial Cell Line‐Derived Neurotrophic Factor in Mesencephalic Neurons
Authors: Sawada Hideyuki , Ibi Masakazu , Kihara Takeshi , Urushitani Makoto , Nakanishi Miki , Akaike Akinori , Shimohama Shun
Published: July 2010

Glial cell line‐derived neurotrophic factor (GDNF) provides neuroprotection, but its neuroprotective mechanism has not been resolved. We investigated the neuroprotective mechanism of GDNF using primary culture of the rat mesencephalon. Bleomycin sulfate (BLM) and L‐buthionine‐[S,R]‐sulfoximine (BSO) caused apoptosis in both dopaminergic and nondopaminergic neurons, as revealed by the presence of chromatin condensation, and positive staining by terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick end‐labeling (TUNEL). GDNF preincubation blocked the neurotoxicity and reduced the number of the TUNEL‐positive cells caused by BLM and BSO exposure. In contrast, GDNF did not provide neuroprotection against glutamate toxicity, which was not accompanied by these apoptotic features. The neuroprotection was mediated by phosphatidylinositol 3‐kinase, an effector downstream from c‐Ret, because it was blocked by LY294002. GDNF pretreatment caused up‐regulation of Bcl‐2 and Bcl‐x. Furthermore, GDNF suppressed oxygen radical accumulation caused by BLM. Apoptosis induced by BLM and BSO was blocked by a caspase‐3 inhibitor. Caspase‐3 activity was elevated by BLM and suppressed by GDNF pretreatment. These findings indicate that GDNF has no effect on necrosis but exerts protection against apoptosis by activation of phosphatidylinositol 3‐kinase and the subsequent up‐regulation of Bcl‐2 and Bcl‐x, which suppresses accumulation of oxygen radicals followed by caspase‐3 activation.
Reduction in TrkA‐Immunoreactive Neurons Is Not Associated with an Overexpression of Galaninergic Fibers Within the Nucleus Basalis in Down's Syndrome
Authors: Sendera T.J. , Ma S.Y. , Jaffar S. , Kozlowski P.B. , Kordower J.H. , Mawal Y. , Saragovi H.U. , Mufson E.J.
Published: July 2010

Down's syndrome (DS) individuals develop neuropathological features similar to Alzheimer's disease (AD), including degeneration of cholinergic basal forebrain (CBF) neurons. In AD a reduction in CBF/trkA‐containing neurons has been suggested to trigger a hyperexpression of galaninergic fibers within the nucleus basalis subfield of the basal forebrain. The present study examined the interrelationship between reductions in CBF/trkA‐containing neurons and the overexpression of galaninergic fibers within the nucleus basalis in DS. Within the nucleus basalis stereologic evaluation revealed a 46% reduction in the number of trkA‐immunopositive neurons, whereas optical density measurements displayed a nonsignificant 18% reduction in neuronal trkA immunoreactivity in DS as compared with age‐matched controls. Western blot analysis also showed a significant reduction in cortical trkA protein levels in DS. A semiquantitative examination of galaninergic fibers in the nucleus basalis revealed only a modest hypertrophy of galaninergic fibers within the nucleus basalis in DS. The present findings indicate a significant reduction in trkA within the nucleus basalis and cortex with only a moderate hypertrophy of galaninergic fibers in DS. These observations suggest that DS may not be an exact genetic model for investigation of changes in the AD basal forebrain.
L‐DOPA Does Not Enhance Hydroxyl Radical Formation in the Nigrostriatal Dopamine System of Rats with a Unilateral 6‐Hydroxydopamine Lesion
Authors: Camp Dianne M. , Loeffler David A. , LeWitt Pater A.
Published: July 2010

The debate about the toxicity of L‐DOPA to dopaminergic neurons has not been resolved. Even though enzymatic and nonenzymatic metabolism of L‐DOPA can produce hydrogen peroxide and oxygen free radicals, there has been controversy as to whether L‐DOPA generates an oxidant stress in vivo. This study determined whether acute or repeated administration of L‐DOPA caused in vivo production of hydroxyl radicals in striatum and other brain regions in rats with a unilateral 6‐hydroxydopamine lesion of the dopaminergic nigrostriatal projections. Salicylate trapping combined with in vivo microdialysis provided measurements of extracellular 2,3‐dihydroxybenzoic acid (2,3‐DHBA) in striatum following L‐DOPA administration systemically (100 mg/kg, i.p.) or by intrastriatal perfusion (1 mM, via the microdialysis probe). Tissue concentrations of 2,3‐DHBA and salicylate were also measured in striatum, ventral midbrain, and cerebellum following repeated administration of L‐DOPA (50 mg/kg, i.p., once daily for 16 days). In each instance, treatment with L‐DOPA did not increase 2,3‐DHBA concentrations, regardless of the nigrostriatal dopamine system's integrity. When added to the microdialysis perfusion medium, L‐DOPA resulted in a significant decrease in the striatal extracellular concentration of 2,3‐DHBA. These results suggest that administration of L‐DOPA, even at high doses, does not induce hydroxyl radical formation in vivo and under some conditions may actually diminish hydroxyl radical activity. Furthermore, prior damage to the nigrostriatal dopamine system does not appear to predispose surviving dopaminergic neurons to increased hydroxyl radical formation following L‐DOPA administration. Unlike L‐DOPA, systemic administration of methamphetamine (10 mg/kg, s.c.) produced a significant increase in the concentration of 2,3‐DHBA in striatal dialysate, suggesting that increased formation of hydroxyl radicals may contribute to methamphetamine neurotoxicity.
ATP‐Sensitive Potassium Channel Regulates Astrocytic Gap Junction Permeability by a Ca2+‐Independent Mechanism
Authors: Velasco Ana , Tabernero Arantxa , Granda Begoña , Medina José M.
Published: July 2010

Using the scrape‐loading technique in cultured astrocytes, we show that sulfonylureas such as tolbutamide and glybenzcyclamide, which inhibit the ATP‐sensitive K+ channel, prevent the inhibition of gap junction permeability caused by several structurally unrelated uncouplers such as oleic acid, arachidonic acid, endothelin‐1, octanol, and α‐glycyrrhetinic acid. When the intracellular level of Ca2+ was diminished, all the uncouplers tested were still able to inhibit gap junction communication, indicating that their inhibitory effect was not mediated by Ca2+. In addition, tolbutamide and glybenzcyclamide prevented the inhibitory effect of these uncouplers in Ca2+‐depleted astrocytes, suggesting that the inhibition of the ATP‐sensitive K+ channel increases gap junction permeability through a Ca2+‐independent mechanism. The activation of the ATP‐sensitive K+ channel caused by potassium channel openers such as diazoxide and pinacidil led to the inhibition of gap junction communication and overcame the effect of sulfonylureas. These results suggest that the ATP‐sensitive K+ channel regulates gap junctional permeability.
Effects of Repeated Antidepressant Treatment on Type 4A Phosphodiesterase (PDE4A) in Rat Brain
Authors: Ye Ying , Jackson Keasha , O'Donnell James M.
Published: July 2010

In a previous study, an up‐regulation of rolipram‐sensitive, low‐Km, cyclic AMP phosphodiesterase (PDE4) subtype PDE4A in rat cerebral cortex following repeated treatment of desipramine was observed. To determine whether this effect is shared by antidepressants from different pharmacological classes, PDE4A expression was examined using immunoblot analyses following repeated treatment with the norepinephrine reuptake inhibitor desipramine, the monoamine oxidase inhibitor phenelzine, the atypical antidepressant trazodone, and the serotonin reuptake inhibitor fluoxetine. Desipramine, phenelzine, and fluoxetine all increased the intensities of the PDE4A bands in hippocampal preparations; trazodone did not. In preparations of cerebral cortex, the intensities of the PDE4A bands were increased following desipramine treatment, not changed following phenelzine or fluoxetine treatment, and decreased following trazodone treatment. It appears that repeated treatment with antidepressant drugs from different pharmacological classes produces similar effects on the expressions of PDE4A variants in hippocampus. This effect is not correlated with the changes in β‐adrenergic receptor densities, suggesting these antidepressants may at some point alter intracellular signal transduction pathways in a similar manner.
Altered G Protein‐Coupling Functions of RNA Editing Isoform and Splicing Variant Serotonin2C Receptors
Authors: Wang Qingde , O'Brien Peter J. , Chen Chun‐Xia , Cho Dan‐Sung C. , Murray John M. , Nishikura Kazuko
Published: July 2010

Different isoforms of serotonin subtype 2C receptor (5‐HT2CR) with altered G protein‐coupling efficacy are generated by RNA editing, which converts genomically encoded adenosine residues into inosines. In combination, editing of five sites all located within the second intracellular loop region of 5‐HT2CR mRNA changes the gene‐encoded Ile, Asn, and Ile at positions 156, 158, and 160, respectively. We analyzed the G protien‐coupling functions of previously unreported editing isoform receptors. An ~13‐fold reduction in the agonist potency for G protein‐coupling stimulation as well as a significantly reduced basal level activity was observed with the thalamus‐specific isoform carrying Ile156, Gly158, and Val160 (5‐HT2CR‐IGV). In contrast, the agonist was four‐ to five‐fold less potent with 5‐HT2CR‐MSV and ‐IDV, detected in the amygdala and choroid plexus, respectively, indicating a dominant role for the amino acid residue at position 158 in receptor functions. We also identified a splicing variant receptor with a truncated C terminus that displayed no ligand binding capacity or G protein‐coupling activity. Examination of the alternatively spliced RNA encoding this truncated receptor suggests that editing of this variant RNA occurs after completion of splicing, resulting in complete editing at all five sites.
Identification of Amino Acid Residues of GABAA Receptor Subunits Contributing to the Formation and Affinity of the tert‐Butylbicyclophosphorothionate Binding Site
Authors: Jursky  , Fuchs  , Buhr  , Tretter  , Sigel  , Sieghart
Published: July 2010

A chimeric GABAA receptor subunit was constructed that contained the β3 sequence from the N‐terminus to the first two amino acids of the second transmembrane (TM2) domain. The remaining part of this chimera had the sequence of the α1 subunit. On co‐expression with α1 subunits, this chimera was able to form heterooligomeric channels that were open in the absence of GABA. Picrotoxin and tert‐butylbicyclophosphorothionate (TBPS) were able to block these channels with low potency. These channels exhibited high‐affinity [3H]muscimol but no high‐affinity [35S]TBPS binding sites. Introduction of V251, A252, and L253 of the β3 subunit into the chimera resulted in the formation of closed channels that could be opened by GABA. The introduction of A252 and L253 of the β3 subunit into this chimera was sufficient to reconstitute the specific high‐affinity [35S]TBPS binding site in receptors composed of the chimera and α1 subunits. Replacement of other amino acids of the TM2 region of the chimera with corresponding amino acids of the β3 subunit modulated the affinity of this [35S]TBPS binding site. Results obtained provide important information on the structure‐function relationship of GABAA receptors.
Effects of l‐dopa and other amino acids against paraquat‐induced nigrostriatal degeneration
Authors: McCormack Alison L. , Di Monte Donato A.
Published: July 2010

Exposure to the herbicide paraquat causes selective nigrostriatal degeneration and aggregation of α‐synuclein in the mouse brain. The purpose of this study was to assess mechanisms of paraquat entry into the CNS and, in particular, the effects of substrates of the blood–brain barrier (BBB) neutral amino acid transporter (System L carrier) on paraquat accumulation and neurotoxicity. Using a paraquat antibody, robust immunoreactivity was observed in the midbrain of mice injected with the herbicide. This immunoreactivity was abolished by administration of l‐valine or l‐phenylalanine, two System L substrates, immediately before paraquat exposure. Pre‐treatment with these amino acids completely protected against paraquat‐induced loss of nigrostriatal dopaminergic cells and formation of thioflavine S‐positive intracellular deposits. Interestingly, the anti‐parkinsonian drug l‐dopa, which is transported across the BBB through the same neutral amino acid carrier, was also neuroprotective when administered 30 min prior to paraquat. In contrast, paraquat‐induced toxicity was unaffected if animals (i) were pre‐treated with d‐valine, the biologically inactive d‐isomer of l‐valine, or with l‐lysine, a substrate of the basic rather than the neutral amino acid carrier, or (ii) were injected with l‐dopa 24 h after paraquat exposure. Data are consistent with a critical role of uptake across the BBB in paraquat neurotoxicity, and suggest that dietary elements (e.g. amino acids) or therapeutic agents (e.g. l‐dopa) may modify the effects of toxicants targeting the nigrostriatal system.
Immunocytochemical localization of glycogen phosphorylase isozymes in rat nervous tissues by using isozyme‐specific antibodies
Authors: Pfeiffer‐Guglielmi Brigitte , Fleckenstein Burkhard , Jung Günther , Hamprecht Bernd
Published: July 2010

Isozyme‐specific antibodies were raised against peptides from the low‐homology regions of the sequences of rat glycogen phosphorylase BB and MM isozymes by immunization of rabbits and guinea pigs. Immunocytochemical double‐labelling experiments on frozen sections of rat nervous tissues were performed to investigate the isozyme localization pattern. Astrocytes throughout the brain and spinal cord expressed both isozymes in perfect co‐localization. Ependymal cells only expressed the BB isozyme. Most neurones were not immunoreactive. The rare neurones that contained glycogen phosphorylase only expressed the BB isozyme. Nearly all of these neurones formed part of the afferent somatosensory system. These findings stress the general importance of glycogen in neural energy metabolism and indicate a special role for the glycogen phosphorylase BB isozyme in neurones in the somatosensory system.