bluuebunny/crossref_metadata_2025 huggingface dataset:
Is Differential Regulation of Mitochondrial Transcripts in Parkinson's Disease Related to Apoptosis?
Authors: Ruberg Merle , Brugg Bernard , Prigent Annick , Hirsch Etienne , Brice Alexis , Agid Yves
Published: July 2010

Abstract: A cDNA library of substantia nigra pars compacta from a patient with Parkinson's disease (PD) was differentially screened with probes of normal and parkinsonian substantia nigra enriched in neuronal transcripts. Fifty‐eight clones were isolated; 39 were subunits of mitochondrial respiratory complexes I and IV. Parallel screening of a cDNA library derived from normal substantia nigra confirmed differential representation of the transcripts in the substantia nigra pars compacta. In situ hybridization in postmortem brain from parkinsonian and control subjects, with representative complex I and complex IV probes, showed increased labeling, at the cellular level, of the complex I subunit ND1 in neurons of the lateral substantia nigra, where cell death is greatest in PD, but decreased labeling in the medial substantia nigra where fewer cells die. Expression of a complex IV subunit, COXI, increased, however, in both parts of the structure. Increased expression of ND1 and COXI was also observed in nerve growth factor‐differentiated PC12 cells undergoing apoptosis induced by tumor necrosis factor‐α, suggesting that the differential regulation of certain mitochondrial mRNAs may be associated with this form of cell death. This in vitro model of apoptosis is potentially relevant to the death of dopaminergic neurons in PD, because these cells express the tumor necrosis factor‐α receptor, and neighboring microglial cells in patients synthesize the cytokine.
Protein Phosphorylation and Calcium Uptake into Rat Forebrain Synaptosomes: Modulation by the σ Ligand, 1,3‐Ditolylguanidine
Authors: Brent Paul J. , Herd Lynn , Saunders Heather , Sim Alistair T. R. , Dunkley Peter R.
Published: July 2010

Abstract: The σ ligand 1,3‐di‐O‐tolylguanidine (DTG) increased basal dynamin and decreased depolarization‐stimulated phosphorylation of the synaptosomal protein synapsin Ib without having direct effects on protein kinases or protein phosphatases. DTG dose‐dependently decreased the basal cytosolic free Ca2+ concentration ([Ca2+]i) and blocked the depolarization‐dependent increases in [Ca2+]i. These effects were inhibited by the σ antagonists rimcazole and BMY14802. The nitric oxide donors sodium nitroprusside (SNP) and 8‐(p‐chlorophenylthio)guanosine‐3′,5′‐cyclic monophosphorothioate decreased basal [Ca2+]i and the KCl‐evoked rise in [Ca2+]i to an extent similar to DTG. SNP, but not DTG, produced a rise in cyclic GMP levels, suggesting that the effect of DTG on [Ca2+]i was not mediated via downstream regulation of cyclic GMP levels. DTG increased 45Ca2+ uptake and efflux under basal conditions and inhibited the 45Ca2+ uptake induced by depolarization with KCl. The KCl‐evoked rise in [Ca2+]i was inhibited by ω‐conotoxin (ω‐CgTx)‐GVIA and ‐MVIIC but not nifedipine and ω‐agatoxin‐IVA. The effect of DTG on decreasing the KCl‐evoked rise in [Ca2+]i was additive with ω‐CgTx‐MVIIC but not with ω‐CgTx‐GVIA. These data suggest that DTG was producing some of its effects on synapsin I and dynamin phosphorylation and intrasynaptosomal Ca2+ levels via inhibition of N‐type Ca2+ channels.
Enhancement of Apoptosis in Developing Chick Neural Retina Cells by Basic Fibroblast Growth Factor
Authors: Yokoyama Yoshiko , Ozawa Satoshi , Seyama Yousuke , Namiki Hideo , Hayashi Yokichi , Kaji Kazuhiko , Shirama Kazuhiko , Shioda Masaki , Kano Kazutaka
Published: July 2010

Abstract: To evaluate the role of various growth factors in naturally occurring cell death during development of the neural retina, we examined the effects of such factors on the nuclear morphology and the size of DNA in cultured chick embryonic neural retina cells. Basic fibroblast growth factor (bFGF) increased internucleosomal cleavage of DNA and nuclear fragmentation in a time‐ and dose‐dependent manner. The effect was inhibited by anti‐bFGF antibody, suramin, and cycloheximide. Epidermal growth factor, platelet‐derived growth factor, nerve growth factor, tumor necrosis factor‐α, and dexamethasone had no effect. These results provide evidence that bFGF may eventually act as a lethal factor inducing apoptotic cell death during the development of the neural retina in chick embryo.
Protein Kinase A Coordinately Regulates Both Basal Expression and Cyclic AMP‐Mediated Induction of Three Catecholamine‐Synthesizing Enzyme Genes
Authors: Hwang Onyou , Park So Yeon , Kim Kwang Soo
Published: July 2010

Abstract: Studies have shown that the cyclic AMP‐regulated pathway is involved in the activation of tyrosine hydroxylase (TH) and in the induction of gene expression of the three catecholamine‐synthesizing enzymes, TH, dopamine β‐hydroxylase (DBH), and phenylethanolamine N‐methyltransferase (PNMT). In the present study we investigated further the role of protein kinase A (PKA) in the regulation of both basal and cyclic AMP‐inducible transcription of the three catecholamine‐synthesizing enzymes in primary cultured bovine chromaffin cells by using the PKA‐specific inhibitor N‐[2‐(p‐bromocinnamylamine)ethyl]‐5‐isoquinolinesulfonamide (H‐89). In the presence of 40 µM H‐89, mRNA levels of TH, DBH, and PNMT were reduced to 17 ± 8, 19 ± 8, and 14 ± 2% of the untreated control, respectively, in 24 h, and intracellular norepinephrine and epinephrine levels were decreased to 20 and 34%, respectively, in 72 h. At 20 µM, although the basal enzyme gene expression levels were little affected, their induction by forskolin was abolished and norepinephrine and epinephrine levels fell to 55 and 74%. This reduction in catecholamines at 20 µM was probably due to changes in the phosphorylation state of TH, as its enzymatic activity was found to be decreased to 66 and 69% in 48 and 72 h, respectively. Thus, PKA activity in bovine adrenal medullary cells coordinately regulates both basal and cyclic AMP‐inducible gene expression of specific catecholamine‐synthesizing enzymes, resulting in changes in intracellular catecholamine levels available for consequent neurohormonal activities.
Molecular Cloning of a Lobster Gαq Protein Expressed in Neurons of Olfactory Organ and Brain
Authors: McClintock Timothy S. , Xu Fuqiang , Quintero Jorge , Gress Anne M. , Landers Teresa M.
Published: July 2010

Abstract: We have isolated from an American lobster (Homarus americanus) olfactory organ cDNA library a clone, hGαq, with >80% identity to mammalian and arthropod Gαq sequences. In brain and olfactory organ, hGαq mRNA was expressed predominantly in neurons, including virtually all the neuronal cell body clusters of the brain. Gαq protein was also expressed broadly, appearing on western blots as a single band of 46 kDa in brain, eyestalk, pereiopod, dactyl, tail muscle, olfactory organ, and aesthetasc hairs. These results suggest that hGαq plays a role in a wide variety of signal transduction events. Its presence in the olfactory aesthetasc hairs, which are almost pure preparations of the outer dendrites of the olfactory receptor neurons, the expression of a single hGαq mRNA species (6 kb) in the olfactory organ, and the localization of hGαq mRNA predominantly in the olfactory receptor neurons of the olfactory organ strongly suggest that one function of hGαq is to mediate olfactory transduction.
Ganglioside GM1 Enhances Induction by Nerve Growth Factor of a Putative Dimer of TrkA
Authors: Farooqui Tahira , Franklin Teresa , Pearl Dennis K. , Yates Allan J.
Published: July 2010

Abstract: GM1 enhances nerve growth factor (NGF)‐stimulated neuritogenesis and prevents apoptotic death of PC12 cells; both may be due to enhancement of TrkA dimerization. In this study, we examined the effect of GM1 on NGF‐induced TrkA dimerization in Trk‐PC12 (6–24) cells. NGF increased tyrosine phosphorylation of the 140‐kDa protein (TrkA monomer), and preincubation with GM1 potentiated this effect. Adding the protein cross‐linker bis(sulfosuccinimidyl) suberate with NGF resulted in the appearance of two major bands (220 and 330 kDa) when probed with antibodies against TrkA or phosphotyrosine, and GM1 also enhanced this effect. We interpret the 330‐kDa band as being a homodimer of TrkA. The identity of the 220‐kDa band is still not certain but may consist of a posttranslationally modified form of TrkA. Our results suggest that GM1 is augmenting the effects of NGF on PC12 cells by enhancing the dimerization and activation of the TrkA receptor.
Time Course Changes in the Dopaminergic Nigrostriatal System Following Transection of the Medial Forebrain Bundle: Detection of Oxidatively Modified Proteins in Substantia Nigra
Authors: Venero José L. , Revuelta Mati , Cano Josefina , Machado Alberto
Published: July 2010

Abstract: We studied the time course of oxidatively modified proteins in the nigrostriatal dopaminergic system following transection of the medial forebrain bundle by quantifying the number of carbonyl groups coupled to striatal and nigral protein homogenates, an index of metal‐catalyzed oxidations. We found a striking effect of axotomy on the number of oxidatively modified proteins in the substantia nigra but not in the striatum within the first 5 days postlesion. This effect was correlated with the neurochemical activity of the dopaminergic and serotoninergic systems in the substantia nigra, which suggests a role of dopamine‐ and serotonin‐derived radical oxygen species in the oxidative stress detected in this brain area. We then searched for the type of cell death in the substantia nigra following axotomy. The fragmentation pattern obtained by agarose gel electrophoresis of DNA isolated from nigral tissue was indicative of cell death being entirely necrotic. In fact, no evidence of apoptosis was detected at any postlesion time as revealed by TdT‐mediated dUTP‐biotin nick end‐labeling (TUNEL) staining. The course of necrotic cell death in the substantia nigra coincided with the maximal levels of oxidatively modified proteins in the substantia nigra, suggesting a link between oxidative stress and nerve cell death and also coinciding with the neurochemical activity of both dopaminergic and serotoninergic systems.
Functional Role of Amino‐Terminal Serine16 and Serine27 of Gαz in Receptor and Effector Coupling
Authors: Ho Maurice K. C. , Wong Yung H.
Published: July 2010

Abstract: The α subunit of Gz (αz) harbors two N‐terminal serine residues (at positions 16 and 27) that serve as protein kinase C‐mediated phosphorylation sites. The cognate residues in the α subunit of Gt1 provide binding surfaces for the β1 subunit. We used three serine‐to‐alanine mutants of αz to investigate the functional importance of the two N‐terminal serine residues. Wild‐type or mutant αz was transiently coexpressed with different receptors and adenylyl cyclase isozymes in human embryonic kidney 293 cells, and agonist‐dependent regulation of cyclic AMP accumulation was examined in a setting where all endogenous α subunits of Gi were inactivated by pertussis toxin. Replacement of one or both serine residues by alanine did not alter the ability of αz to interact with δ‐opioid, dopamine D2, or adenosine A1 receptors. Its capacity to inhibit endogenous and type VI adenylyl cyclases was also unaffected. Functional release of βγ subunits from the mutant αz subunits was not impaired because they transduced βγ‐mediated stimulation of type II adenylyl cyclase. Constitutively active mutants of all four αz subunits were constructed by the introduction of a Q205L mutation. The activated mutants showed differential abilities to inhibit human choriogonadotropin‐mediated cyclic AMP accumulation in luteinizing hormone receptor‐transfected cells. Loss of both serine residues, but not either one alone, impaired the receptor‐independent inhibition of adenylyl cyclase by the GTPase‐deficient mutant. Thus, replacement of the amino‐terminal serine residues of αz has no apparent effect on receptor‐mediated responses, but these serine residues may be essential for ensuring transition of αz into the active conformation.
Dinucleotide Receptor Modulation by Protein Kinases (Protein Kinases A and C) and Protein Phosphatases in Rat Brain Synaptic Terminals
Authors: Pintor Jesús , Gualix Javier , Miras‐Portugal M. Teresa
Published: July 2010

Abstract: The diadenosine polyphosphates, diadenosine tetraphosphate and diadenosine pentaphosphate (Ap5A), can activate an ionotropic dinucleotide receptor that induces Ca2+ transients into synaptosomes prepared from rat brain. This receptor, also termed the P4 purinoceptor, is sensitive only to adenine dinucleotides and is insensitive to ATP. Studies on the modulatory role of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatases on the response of diadenosine polyphosphate receptors were performed by measuring the changes in the intracellular Ca2+ levels with fura‐2. Activation and inhibition of PKA were carried out by means of forskolin and the PKA inhibitory peptide (PKA‐IP), respectively. The Ap5A response was inhibited by forksolin to 35% of control values, but PKA‐IP induced an increase of 37%. The effect of PKC activation was similar to that observed for PKA. PKC stimulation with phorbol 12,13‐dibutyrate produced an inhibition of 67%, whereas the PKC inhibitors staurosporine and PKC inhibitory peptide enhanced the responses elicited by Ap5A to 40% in both cases. Protein phosphatase inhibitors diminished the responses elicited by Ap5A to 17% in the case of okadaic acid, to 50% for microcystin, and to 45% in the case of cyclosporin A. Thus, the activity of dinucleotide receptors in rat brain synaptosomes appears to be modulated by phosphorylation/dephosphorylation. These processes could be of physiological significance in the control of transmitter release from neurons that are postsynaptic to nerves that release diadenosine polyphosphates.
α2,6‐Sialyltransferase Gene Transfection into a Human Glioma Cell Line (U373 MG) Results in Decreased Invasivity
Authors: Yamamoto Hirotaka , Kaneko Yoichi , Rebbaa Abdelhadi , Bremer Eric G. , Moskal Joseph R.
Published: July 2010

Abstract: Glycosyltransferase gene transfection into cell lines has been an approach used successfully to elucidate the functional role of cell surface glycoconjugates. We have transfected the rat CMP‐NeuAc:Galβ1,4GlcNAc α2,6‐sialyltransferase (EC 2.4.99.1) gene into a human, tumorigenic, glioma cell line, U373 MG. This transfection led to a marked inhibition of invasivity, alterations in adhesivity to fibronectin and collagen matrices, and inappropriately sialylated α3β1 integrin. Adhesion‐mediated protein tyrosine phosphorylation was reduced in the transfectants despite increased expression of focal adhesion kinase, p125fak. Furthermore, the transfectants showed a distinct cell morphology, an increased number of focal adhesion sites, and different sensitivity to cytochalasin D treatment than control U373 MG cells. These results suggest that inappropriate sialylation of cell surface glycoconjugates, such as integrins, can change focal adhesion as well as adhesion‐mediated signal transduction and block glioma cell invasivity in vitro.
Site‐Directed Deletion of a 10‐Nucleotide Domain of the 3′‐Untranslated Region of the GLUT1 Glucose Transporter mRNA Eliminates Cytosolic Protein Binding in Human Brain Tumors and Induction of Reporter Gene Expression
Authors: Tsukamoto Haruhisa , Boado Ruben J. , Pardridge William M.
Published: July 2010

Abstract: The posttranscriptional regulation of GLUT1 glucose transporter gene expression may be mediated by specific interactions between cytosolic trans‐acting factors and regulatory cis‐elements within the 3′‐untranslated regions (UTRs) of the GLUT1 mRNA. Recent studies demonstrate that experimental and human brain tumors express an 80‐kDa protein that reacts with a specific sequence around nucleotide 2,200 within the GLUT1 mRNA 3′‐UTR. The 80‐kDa protein is selectively expressed in hemangioblastoma, a tumor characterized by overexpression of GLUT1. The enhancer role of this GLUT1 3′‐UTR cis‐element was confirmed in the present studies using the luciferase expression vector pGL2 and site‐directed deletion. Transfection of C6 glioma cells with pGL2 (containing nucleotides 2,100–2,300 of the bovine GLUT1 3′‐UTR inserted at the PflMI site within the luciferase 3′‐UTR) results in a fivefold increase in luciferase gene expression. Deletion of nucleotides 2,181–2,190 of the bovine GLUT1 3′‐UTR, i.e., the putative binding site of the 80‐kDa protein, completely eliminated the enhancement of luciferase activity in the transfected cells. Luciferase mRNA containing the putative cis‐element inserted in the 3′‐UTR was transcribed, and after UV cross‐linking, this mRNA complexed with the 80‐kDa protein in cytosol of either C6 cells or hemangioblastoma. In contrast, this complex was undetected with either luciferase control mRNA or 10 nucleotide‐deleted RNA. The present study provides evidence that nucleotides 2,181‐2,190 of the bovine GLUT1 mRNA 3′‐UTR forms a complex with brain tumor cytosolic proteins that serves to increase GLUT1 gene expression at the posttranscriptional level.
ICE/CED‐3 Family Executes Oligodendrocyte Apoptosis by Tumor Necrosis Factor
Authors: Hisahara Shin , Shoji Shin'ichi , Okano Hideyuki , Miura Masayuki
Published: July 2010

Abstract: Tumor necrosis factor (TNF) is thought to be one of the mediators responsible for the damage of oligodendrocytes (OLGs) in multiple sclerosis (MS). We report here the involvement of the interleukin 1β‐converting enzyme (ICE)/Caenorhabditis elegans gene ced‐3 (CED‐3) family in TNF‐mediated cell death of OLGs. The addition of TNF‐α to primary cultures of OLGs that express ice and cpp32 significantly decreased the number of live OLGs in 72 h. DNA fragmentation was detected in TNF‐treated OLGs at 36 h with the terminal deoxynucleotidyl transferase dUTP nick end‐labeling assay. Benzyloxycarbonyl‐Asp‐CH2OC(O)‐2,6‐dichlorobenzene, an inhibitor of the ICE/CED‐3 family that shows p35‐like inhibitory specificity, protected against the TNF‐induced cell death of OLGs. Furthermore, acetyl‐YVAD‐CHO (a specific inhibitor of ICE‐like proteases) as well as acetyl‐DEVD‐CHO (a specific inhibitor of CPP32‐like proteases) enhanced the survival of OLGs treated with TNF‐α, indicating that ICE‐ and the CPP32‐mediated cell death pathways are activated in TNF‐induced OLG cell death. Our results suggest that the inhibition of ICE/CED‐3 proteases may be a novel approach to treat neurodegenerative diseases such as MS.
Isoform‐Specific Modulation by Apolipoprotein E of the Activities of Secreted β‐Amyloid Precursor Protein
Authors: Barger Steven W. , Mattson Mark P.
Published: July 2010

Abstract: The genes for both the β‐amyloid precursor protein and apolipoprotein E (ApoE) have been linked to Alzheimer's disease. This connection suggests the possibility that these proteins interact physically or functionally. To explore this idea, we focused on the neuroprotective activity of secreted amyloid precursor protein (sAPP) and related signal transduction events. After coincubation with ApoE, sAPP exhibited an enhanced [Ca2+]i‐lowering activity and enhanced protection against excitotoxicity in rat primary hippocampal neurons. In contrast, the stimulation of phosphoinositide production by sAPP was inhibited by ApoE. Kinetic analyses and coimmunoprecipitation experiments indicated that these actions result from formation of a heteromeric complex between ApoE and sAPP. Furthermore, the ApoE4 isoform, which seems to accelerate the onset of Alzheimer's disease, was less potent than ApoE3 in modifying each activity of sAPP. These data suggest that sAPP‐dependent neuroprotective mechanisms would be compromised in individuals expressing ApoE4, a scenario that may contribute to the development of Alzheimer's disease.
Construction and Characterization of Ciliary Neurotrophic Factor (CNTF) Antagonists: Microenvironmental Difference in the CNTF Receptor Between Rat and Chicken Cells for Recognizing the D1 Cap Region
Authors: Inoue Makoto , Karita Hiromi , Nakayama Chikao , Noguchi Hiroshi
Published: July 2010

Abstract: Antagonistic mutants of ciliary neurotrophic factor (CNTF) were constructed and their properties characterized. K155A and K155W mutants lost cell survival promoting activity for chicken dorsal root ganglion (DRG) neurons and inhibited the activity of the wild type. However, they retained slight agonistic activity for the survival of rat DRG neurons, indicating there is a difference between chicken and rat cells for receptor recognition around the D1 cap region including K155 residue. The chicken receptor recognizes the D1 cap region more strictly than does the rat receptor. The substitution of F152, which locates at the top of the D1 cap region, was combined with the K155A mutation. A combination of the two mutations gave an antagonistic feature to not only chicken but also rat cells. Both F152S/K155A and F152D/K155A mutants lacked cell survival promoting activity and had an antagonistic effect on rat DRG neurons. The three‐dimensional structure of CNTF suggests the following. F152 and K155 bind to the receptor with hydrophobic and electrostatic interactions, respectively. F152 locates close to L156 with a van der Waals contact, and K155 contacts with Q42 through a hydrogen bond. Both interactions play indispensable roles in maintaining the structure around the D1 cap region of CNTF.
Ganglioside GM1 Activates the Mitogen‐Activated Protein Kinase Erk2 and p70 S6 Kinase in U‐1242 MG Human Glioma Cells
Authors: Van Brocklyn James R. , Vandenheede J. R. , Fertel Richard , Yates Allan J. , Rampersaud Arfaan A.
Published: July 2010

Abstract: Gangliosides are implicated in the regulation of cellular proliferation as evidenced by differences in ganglioside composition associated with malignant transformation and density of cells in culture, as well as their inhibitory effects when added to cells growing in culture. Exogenously added gangliosides have a bimodal effect on proliferation in U‐1242 MG glioma cells, inhibiting DNA synthesis in growing cells and stimulating it in quiescent cells. We investigated the mechanisms involved in stimulation of DNA synthesis using [3H]thymidine incorporation and immune complex kinase assays to identify responsible signal transduction pathways. Treatment of quiescent U‐1242 MG cells with GM1 caused activation of the mitogen‐activated protein (MAP) kinase isoform Erk2. Pretreatment with the specific MAP kinase kinase inhibitor PD98059 prevented the GM1‐stimulated Erk2 activation and GM1‐stimulated DNA synthesis. GM1 treatment stimulated another distinct signaling pathway leading to activation of p70 S6 kinase (p70s6k), and this was prevented by pretreatment with rapamycin. Rapamycin also inhibited GM1‐stimulated DNA synthesis. Activation of both pathways and stimulation of DNA synthesis were inhibited by forskolin treatment; however, GM1 had no effect on cyclic AMP levels. Platelet‐derived growth factor also activated both Erk2 and p70s6k but did not cause DNA synthesis, suggesting that GM1 may stimulate additional cascades, which also contribute to GM1‐mediated DNA synthesis.
Metabotropic Glutamate Receptor mGluR5 in Astrocytes: Pharmacological Properties and Agonist Regulation
Authors: Balázs Robert , Miller Stephan , Romano Carmelo , De Vries Annick , Chun Yong , Cotman Carl W.
Published: July 2010

Abstract: Metabotropic glutamate receptor (mGluR) agonists induce extensive phosphoinositide (PI) hydrolysis in astrocytes grown in a chemically defined medium with select growth factors. These astrocytes express mGluR5 transcripts, but none of the splice variants of mGluR1, thus permitting the characterization of mGluR5 in a native CNS cell without interference from mGluR1 activity. mGluR5 activation (1) was not associated with stimulation of cyclic AMP formation, (2) showed high sensitivity to the removal of extracellular versus intracellular Ca2+, (3) displayed high coupling efficiency relative to receptor density, and (4) induced PI hydrolysis that was suppressed by phorbol esters with low potency. The rank order of agonist potency was similar to that observed in mGluR1 and mGluR5 transfected cells. The phenylglycine antagonists tested were effective in blocking responses to 1‐aminocyclopentane‐1S,3R‐dicarboxylic acid, but not to glutamate. Prolonged exposure to agonists induced a two‐phase desensitization of mGluR5 function, an initial phase (completed by 1 h and plateaus for another 3 h) and a late phase (progressive decrease to ∼30% of control levels by 24 h). Only the latter phase was associated with receptor down‐regulation. Desensitization of mGluR5 function did not involve receptor internalization or phosphorylation mediated by protein kinase C or A; it was purely homologous, and reversible. Resensitization after short agonist treatment did not require prior receptor sequestration. Recovery after prolonged agonist exposure required new protein synthesis, but the restoration of function was more rapid than normalization of receptor protein levels, indicating that regulation also involves other components of the transduction system. The protracted desensitization of mGluR5 in astrocytes suggests that the functions mediated by this receptor are maintained under a variety of conditions ranging from repetitive stimulation to injury responses.
BIMG 80, a Novel Potential Antipsychotic Drug: Evidence for Multireceptor Actions and Preferential Release of Dopamine in Prefrontal Cortex
Authors: Volonté Marina , Monferini Eugenia , Cerutti Monica , Fodritto Fabio , Borsini Franco
Published: July 2010

Abstract: In radioligand binding studies, BIMG 80, a new putative antipsychotic, displayed good affinity at certain serotonin (5‐HT1A, 5‐HT2A, 5‐HT6), dopamine (D1, D2L, D4), and noradrenergic (α1) receptors. The effect of acute subcutaneous BIMG 80, clozapine, haloperidol, risperidone, amperozide, olanzapine, and Seroquel was then investigated on dopamine release in medial prefrontal cortex, nucleus accumbens, and striatum in freely moving rats using the microdialysis technique. Four different neurochemical profiles resulted from the studies: (a) Systemic administration of BIMG 80, clozapine, and amperozide produced greater percent increases in dopamine efflux in medial prefrontal cortex than in the striatum or the nucleus accumbens. (b) Haloperidol induced a similar increase in dopamine concentrations in the striatum and nucleus accumbens with no effect in the medial prefrontal cortex. (c) Risperidone and olanzapine stimulated dopamine release to a similar extent in all brain regions investigated. (d) Seroquel failed to change significantly dopamine output both in the medial prefrontal cortex and in the striatum. Because an increase in dopamine release in the medial prefrontal cortex may be predictive of effectiveness in treating negative symptoms and in the striatum may be predictive of induction of extrapyramidal side effects, BIMG 80 appears to be a potential antipsychotic compound active on negative symptoms of schizophrenia with a low incidence of extrapyramidal side effects.
Reactivating Kinase/p38 Phosphorylates τ Protein In Vitro
Authors: Reynolds C. Hugh , Nebreda Angel R. , Gibb Graham M. , Utton Michelle A. , Anderton Brian H.
Published: July 2010

Abstract: Neurofibrillary tangles, one of the major pathological hallmarks of Alzheimer‐diseased brains, consist primarily of aggregated paired helical filaments (PHFs) of hyperphosphorylated τ protein. τ from normal brain and especially from foetal brain is also phosphorylated on some of the sites phosphorylated in PHFs, mainly at serines or threonines followed by prolines. A number of protein kinases can phosphorylate τ in vitro; those that require or accept prolines include GSK3 and members of the mitogen‐activated protein (MAP) kinase family, ERK1, ERK2, and SAP kinase‐β/JNK. In this report, we show that another member of the MAP kinase family, the stress‐activated kinase p38/RK, can phosphorylate τ in vitro. Western blots with phosphorylation‐sensitive antibodies showed that p38, like ERK2 and SAP kinase‐β/JNK, phosphorylated τ at sites found phosphorylated physiologically (Thr181, Ser202, Thr205, and Ser396) and also at Ser422, which is phosphorylated in neurofibrillary tangles but not in normal adult or foetal brain. These findings support the possibility that cellular stress might contribute to τ hyperphosphorylation during the formation of PHFs, and hence, to the development of τ pathology.
Loss of the Xeroderma Pigmentosum Group A Gene (XPA) Enhances Apoptosis of Cultured Cerebellar Neurons Induced by UV but Not by Low‐K+ Medium
Authors: Enokido Yasushi , Inamura Naoko , Araki Toshiyuki , Satoh Takumi , Nakane Hironobu , Yoshino Masafumi , Nakatsu Yoshimichi , Tanaka Kiyoji , Hatanaka Hiroshi
Published: July 2010

Abstract: To study the involvement of the xeroderma pigmentosum group A gene (XPA) in neuronal apoptosis, we cultured cerebellar neurons from mice lacking XPA gene (XPA−/−) and induced apoptosis by exposure to UV irradiation or medium containing a low concentration of potassium (low‐K+ medium). When cerebellar neurons from postnatal days 15–16 wild‐type mice were treated with UV irradiation, apoptotic neuronal death was observed after 24–48 h. About 60% of neurons survived 48 h after UV irradiation at a dose of 5 J/m2. On the other hand, neurons from XPA−/− mice showed a significantly increased vulnerability to UV irradiation, and >90% of neurons died 48 h after UV irradiation at a dose of 5 J/m2. In contrast, low‐K+ medium induced apoptosis of neurons from mice of each genotype with the same kinetics. These results suggest that the XPA gene is involved in neuronal DNA repair and that it thereby influences apoptosis induced by DNA damage in cultured cerebellar neurons.
S‐Adenosylmethionine Decarboxylase Activity Is Decreased in the Rat Cortex After Traumatic Brain Injury
Authors: Henley Charles M. , Wey Karen , Takashima Amy , Mills Charles , Granmayeh Elaine , Krishnappa Indra , Robertson Claudia S.
Published: July 2010

Abstract: S‐Adenosyl‐l‐methionine decarboxylase (SAMdc) and l‐ornithine decarboxylase (ODC) are major enzymes regulating polyamine synthesis. Following ischemia, putrescine content increases as a result of post‐traumatic activation of ODC and inhibition of SAMdc. These alterations are thought to mediate edema and cell death. The purpose of this study was to quantify SAMdc activity and edema in the brain following controlled cortical impact injury. Anesthetized adult male rats underwent a right parietal craniectomy and were subjected to cortical impact injury. Tissues were obtained from three bilateral regions: parietal cortex, motor area (CPm); parietal cortex, somatosensory area (CPs); and the pyriform cortex (CPF). SAMdc activity was determined in the postmitochondrial fraction from homogenates of fresh, unfrozen tissues by measuring the decarboxylation of S‐adenosyl‐l‐[carboxyl‐14C]methionine. Basal SAMdc activity was determined in unoperated rats, and regional differences were noted: Activity was lower in the CPF than in the CPm and CPs. SAMdc activity decreased to the greatest extent in the ipsilateral CPm (impact site) from 1 to 72 h following traumatic brain injury. Significant edema was found in the ipsilateral CPm 1, 8, 16, 24, and 48 h after injury. Decreased SAMdc activity impairs the conversion of putrescine to polyamines and may contribute to delayed pathological changes in the brain after traumatic injury.
Iodoacetate Produces Striatal Excitotoxic Lesions
Authors: Matthews Russell T. , Ferrante Robert J. , Jenkins Bruce G. , Browne Susan E. , Goetz Katrine , Berger Stephanie , Chen Iris Y.‐C. , Beal M. Flint
Published: July 2010

Abstract: Impairment of energy production may play a role in the pathogenesis of Huntington's disease (HD). It was recently shown that huntingtin can bind to and possibly inhibit the glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). We found that intrastriatal administration of the GAPDH inhibitor iodoacetate produces striatal lesions that are significantly attenuated by removal of the corticostriatal glutamatergic input, consistent with an excitotoxic mechanism. The lesions are accompanied by increased production of hydroxyl free radicals as assessed by conversion of salicylate to 2,3‐ and 2,5‐dihydroxybenzoic acid. In vivo magnetic resonance imaging showed lesions on T2‐weighted scans, but there was only a small increase in lactate content. These results show that inhibition of GAPDH produces striatal lesions in vivo and suggest that inhibition of GAPDH could contribute to neuronal degeneration in HD.
A Novel cdc2‐Related Protein Kinase Expressed in the Nervous System
Authors: Lazzaro Maribeth A. , Albert Paul R. , Julien Jean‐Pierre
Published: July 2010

Abstract: We report the cloning and characterization of a cDNA encoding a cdc2‐related protein kinase, named PFTAIRE, that is expressed primarily in the postnatal and adult nervous system. We have demonstrated by in situ hybridization and indirect immunofluorescence that several populations of terminally differentiated neurons and some neuroglia expressed PFTAIRE mRNA and protein. In neurons, PFTAIRE protein was localized in the nucleus and cytoplasm of cell bodies. The anatomical, cellular, and ontogenic patterns of PFTAIRE expression in the nervous system differed from those of p34cdc2 and cdk5, which are expressed in brain and several other mitotic tissues. Proteins of ∼58–60 kDa coprecipitated specifically with PFTAIRE from cytosolic protein preparations of adult mouse brain and transfected cells. These proteins appeared to be the major endogenous substrates associated with this kinase activity. The temporal and spatial expression patterns of PFTAIRE in the postnatal and adult nervous system suggest that PFTAIRE kinase activity may be associated with the postmitotic and differentiated state of cells in the nervous system and that its function may be distinct from those of p34cdc2 and cdk5.
In Vivo Evidence that GABAB Receptors Are Negatively Coupled to Adenylate Cyclase in Rat Striatum
Authors: Hashimoto Tsuneichi , Kuriyama Kinya
Published: July 2010

Abstract: The characteristics of the cerebral GABAB receptor/cyclic AMP (cAMP)‐generating system were investigated using the in vivo microdialysis technique in freely moving rats. Addition of forskolin, an activator of adenylate cyclase, to perfusate for 20 min resulted in a dose‐dependent increase of cAMP efflux from the striatum. Pre‐ and coinfusions of baclofen for 80 min had no effect on the basal efflux of cAMP from the striatum but induced a significant decrease of forskolin (10 µM)‐stimulated cAMP efflux from the striatum in a dose‐dependent manner. SKF 97541 (100 µM), a GABAB receptor agonist, and GABA (50 µM) also decreased forskolin‐induced cAMP efflux from the striatum. Coinfusion of CGP 54626A (100 µM), a GABAB receptor antagonist, counteracted the effect of baclofen on the forskolin‐stimulated cAMP efflux. In contrast, the isoproterenol (5 mM)‐induced increase of cAMP efflux from the striatum was significantly enhanced by pre‐ and coinfusions with baclofen. These results suggest that this test system using in vivo microdialysis may be useful for examining the effect of drugs on the GABAB receptor‐linked cAMP‐generating system in vivo.
Brain Lactate Is an Obligatory Aerobic Energy Substrate for Functional Recovery After Hypoxia: Further In Vitro Validation
Authors: Schurr Avital , Payne Ralphiel S. , Miller James J. , Rigor Benjamin M.
Published: July 2010

Abstract: This study used the rat hippocampal slice preparation and the monocarboxylate transporter inhibitor, α‐cyano‐4‐hydroxycinnamate (4‐CIN), to assess the obligatory role that lactate plays in fueling the recovery of synaptic function after hypoxia upon reoxygenation. At a concentration of 500 µM, 4‐CIN blocked lactate‐supported synaptic function in hippocampal slices under normoxic conditions in 15 min. The inhibitor had no effect on glucose‐supported synaptic function. Of control hippocampal slices exposed to 10‐min hypoxia, 77.8 ± 6.8% recovered synaptic function after 30‐min reoxygenation. Of slices supplemented with 500 µM 4‐CIN, only 15 ± 10.9% recovered synaptic function despite the large amount of lactate formed during the hypoxic period and the abundance of glucose present before, during, and after hypoxia. These results indicate that 4‐CIN, when present during hypoxia and reoxygenation, blocks lactate transport from astrocytes, where the bulk of anaerobic lactate is formed, to neurons, where lactate is being utilized aerobically to support recovery of function after hypoxia. These results unequivocally validate that brain lactate is an obligatory aerobic energy substrate for posthypoxia recovery of function.
Demonstration of an E‐box and Its CNS‐Related Binding Factors for Transcriptional Regulation of the Mouse Type 1 Inositol 1,4,5‐Trisphosphate Receptor Gene
Authors: Konishi Yoshiyuki , Kobayashi Yasushi , Kishimoto Toshihiko , Makino Yasutaka , Miyawaki Atsushi , Furuichi Teiichi , Okano Hideyuki , Mikoshiba Katsuhiko , Tamura Taka‐aki
Published: July 2010

Abstract: The type 1 inositol 1,4,5‐trisphosphate receptor (IP3R1) is expressed abundantly in the CNS, such as in cerebellar Purkinje cells and the hippocampus. We established a tissue‐specific cell‐free transcription system and studied regulatory properties of the 5′ upstream region of the IP3R1 gene by use of this system. Deletion analyses of the promoter revealed several cis elements that function significantly in brain nuclear extracts. Among those elements, sequences from −398 to −295 showed the most predominant cerebellum‐specific positive function. Footprint analyses demonstrated a factor‐binding region from −334 to −318, termed box‐I, that contained an E‐box consensus sequence. Electrophoretic mobility shift assay revealed CNS‐related basic helix‐loop‐helix proteins for the box‐I. Mutational studies using the function assay and competitive electrophoretic mobility shift assays demonstrated a good correlation between the box‐I‐binding factors and the activated transcription. Box‐I‐binding factors were present abundantly in adult mouse CNS, whereas their existence was restricted in embryonic and nonneural tissues. Transient chloramphenicol acetyltransferase assay for the IP3R1 promoter revealed the requirement of box‐I in Neuro2a neuroblastoma cells. In the postnatal CNS, multiple basic helix‐loop‐helix factors are expressed abundantly, some of which are suggested to activate IP3R1 gene expression in the mammalian CNS.
Apoptosis of Retinal Photoreceptors During Development In Vitro: Protective Effect of Docosahexaenoic Acid
Authors: Rotstein Nora P. , Aveldaño Marta I. , Barrantes Francisco J. , Roccamo Ana M. , Politi Luis E.
Published: July 2010

Abstract: When rat retinal cells are cultured in a serum‐free medium, the photoreceptor cells start dying after 7 days. The addition of docosahexaenoic acid (DHA) to the cultures prevents the selective death of photoreceptors. Here it is shown that, unlike other retinal neurons, photoreceptors die through an apoptotic pathway. Hallmarks of apoptosis, such as nuclear fragmentation and condensation and DNA cleavage forming a ladder pattern on an agarose gel, were observed. The timing and high selectivity of the triggering of photoreceptor cell apoptosis suggest the existence of a programmed cell death. Compared with other fatty acids, DHA not only was the most effective in promoting photoreceptor survival, but also the only one to decrease the number of apoptotic nuclei. The results suggest that DHA is important among the factors preventing apoptosis of photoreceptors in the developing retina. A limitation in the availability of this fatty acid might trigger apoptosis as a result of the failure to develop functional photoreceptor outer segments.
Microtubule Dynamics Regulates the Level of Endothelin‐B Receptor in Rat Cultured Astrocytes
Authors: Ozawa Fujiko , Kasuya Yoshitoshi , Hama Hiroshi , Asada Sachie , Inada Tetsuo , Goto Katsutoshi
Published: July 2010

Abstract: We investigated the effect of cytoskeleton modulators on endothelin‐B (ETB) receptor expression in rat primary cultured astrocytes. Northern blot analysis and a binding study revealed that colchicine and nocodazole, microtubule‐disrupting agents, decreased the levels of both ETB receptor mRNA and the number of ET‐1 binding sites in quiescent astrocytes. Down‐regulation of both ETB receptor mRNA and the number of binding sites for ET‐1 was also observed in quiescent astrocytes treated with taxol, a microtubule‐stabilizing agent. In contrast, neither β‐lumicolchicine, an inactive isomer of colchicine, nor cytochalasin D, a microfilament‐disrupting agent, influenced ETB receptor expression. The level of ETB receptors in astrocytes was affected by the cell state, namely, proliferative, quiescent, or differentiated state. The order of ETB receptor expression according to the cell state was proliferative state < quiescent state ≪ differentiated state induced by dibutyryl cyclic AMP. Also, in proliferative astrocytes and differentiated astrocytes, colchicine significantly down‐regulated both ETB receptor mRNA and the number of binding sites for ET‐1. However, thymidine assay revealed that colchicine did not change quiescent astrocytes and differentiated astrocytes to a proliferative state. Furthermore, the increase in glutamine synthetase activity in differentiated astrocytes was not affected by colchicine. These results suggest that microtubule dynamics possibly regulates ETB receptor expression in astrocytes without affecting the cell state.
4‐Hydroxynonenal, an Aldehydic Product of Lipid Peroxidation, Impairs Signal Transduction Associated with Muscarinic Acetylcholine and Metabotropic Glutamate Receptors: Possible Action on Gαq/11
Authors: Blanc Emmanuelle M. , Kelly Jeremiah F. , Mark Robert J. , Waeg Georg , Mattson Mark P.
Published: July 2010

Abstract: Considerable data indicate that oxidative stress and membrane lipid peroxidation contribute to neuronal degeneration in an array of age‐related neurodegenerative disorders. In contrast, the impact of subtoxic levels of membrane lipid peroxidation on neuronal function is largely unknown. We now report that 4‐hydroxy‐nonenal (HNE), an aldehydic product of lipid peroxidation, disrupts coupling of muscarinic cholinergic receptors and metabotropic glutamate receptors to phospholipase C‐linked GTP‐binding proteins in cultured rat cerebrocortical neurons. At subtoxic concentrations, HNE markedly inhibited GTPase activity, inositol phosphate release, and elevation of intracellular calcium levels induced by carbachol (muscarinic agonist) and (RS)‐3,5‐dihydroxyphenyl glycine (metabotropic glutamate receptor agonist). Maximal impairment of agonist‐induced responses occurred within 30 min of exposure to HNE. Other aldehydes, including malondialdehyde, had little effect on agonist‐induced responses. Antioxidants that suppress lipid peroxidation did not prevent impairment of agonist‐induced responses by HNE, whereas glutathione, which is known to bind and detoxify HNE, did prevent impairment of agonist‐induced responses. HNE itself did not induce oxidative stress. Immunoprecipitation‐western blot analysis using an antibody to HNE‐protein conjugates showed that HNE can bind to Gαq/11. HNE also significantly suppressed inositol phosphate release induced by aluminum fluoride. Collectively, our data suggest that HNE plays a role in altering receptor‐G protein coupling in neurons under conditions of oxidative stress that may occur both normally, and before cell degeneration and death in pathological settings.
Mechanism of Cellular 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) Reduction
Authors: Liu Yuanbin , Peterson Daniel A. , Kimura Hideo , Schubert David
Published: July 2010

Abstract: 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction is one of the most frequently used methods for measuring cell proliferation and neural cytotoxicity. It is widely assumed that MTT is reduced by active mitochondria in living cells. By using isolated mitochondria from rat brain and B12 cells, we indeed found that malate, glutamate, and succinate support MTT reduction by isolated mitochondria. However, the data presented in this study do not support the exclusive role of mitochondria in MTT reduction by intact cells. Using a variety of approaches, we found that MTT reduction by B12 cells is confined to intracellular vesicles that later give rise to the needle‐like MTT formazan at the cell surface. Some of these vesicles were identified as endosomes or lysosomes. In addition, MTT was found to be membrane impermeable. These and other results suggest that MTT is taken up by cells through endocytosis and that reduced MTT formazan accumulates in the endosomal/lysosomal compartment and is then transported to the cell surface through exocytosis.
Extracellular Calcium Sensing by Glial Cells: Low Extracellular Calcium Induces Intracellular Calcium Release and Intercellular Signaling
Authors: Zanotti Stephen , Charles Andrew
Published: July 2010

Abstract: Glial cells in primary mixed cultures or purified astrocyte cultures from mouse cortex respond to reduced extracellular calcium concentration ([Ca2+]e) with increases in intracellular calcium concentration ([Ca2+]i) that include single‐cell Ca2+ oscillations and propagated intercellular Ca2+ waves. The rate and pattern of propagation of low [Ca2+]e‐induced intercellular Ca2+ waves are altered by rapid perfusion of the extracellular medium, suggesting the involvement of an extracellular messenger in Ca2+ wave propagation. The low [Ca2+]e‐induced Ca2+ response is abolished by thapsigargin and by the phospholipase antagonist U73122. The low [Ca2+]e‐induced response is also blocked by replacement of extracellular Ca2+ with Ba2+, Zn2+, or Ni2+, and by 100 µM La3+. Glial cells in lowered [Ca2+]e(0.1–0.5 mM) show an increased [Ca2+]i response to bath application of ATP, whereas glial cells in increased [Ca2+]e (10–15 mM) show a decreased [Ca2+]i response to ATP. These results show that glial cells possess a mechanism for coupling between [Ca2+]e and the release of Ca2+ from intracellular stores. This mechanism may be involved in glial responses to the extracellular environment and may be important in pathological conditions associated with low extracellular Ca2+ such as seizures or ischemia.
Regulation of Astrocyte Nitric Oxide Synthase Type II Expression by ATP and Glutamate Involves Loss of Transcription Factor Binding to DNA
Authors: Lin Hsin Lee , Murphy Sean
Published: July 2010

Abstract: Pretreatment of astrocytes with either glutamate or ATP, acting via specific receptors, suppresses subsequent cytokine‐induced expression of type II nitric oxide synthase (NOS). This effect is downstream from the activation and translocation of nuclear factor (NF)‐κB, as western blotting revealed no difference in accumulation of the p50 subunit in nuclear extracts from agonist‐treated vs. nontreated cells. However, evidence from gel‐shift assay suggests that the binding of nuclear protein from agonist‐treated cells to NF‐κB and activator protein‐1 consensus oligonucleotides is markedly reduced. Selective inhibitors of protein kinases A and C could not restore either binding of transcription factors to DNA or type II NOS mRNA expression in agonist‐pretreated cells. The modulation of proinflammatory cytokine‐evoked type II NOS expression by ATP and glutamate may play an important role in CNS pathologies associated with stroke and trauma.