bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Cloning and Expression of a Mouse Brain cDNA Encoding a Novel Protein Target of Calcyclin
Authors: Filipek Anna , Kuźnicki Jacek
Published: July 2010

Abstract: A protein target of mouse calcyclin, p30, which we call calcyclin‐binding protein (CacyBP), was identified in mouse brain and Ehrlich ascites tumor (EAT) cells. The amino acid sequence of the CacyBP chymotryptic peptide was used to prepare synthetic oligonucleotides that served as a probe to screen the mouse brain cDNA library. A 1.4‐kb positive clone was detected, isolated, and sequenced. The analyzed clone contains an open reading frame encoding a protein of a molecular mass of ∼26 kDa. The nucleotide and predicted amino acid sequences indicate that CacyBP is a novel protein. The results obtained from northern blots show that the CacyBP gene is expressed predominantly in mouse brain and EAT cells. Using a pGEX vector the recombinant CacyBP was expressed in Escherichia coli, and its properties were analyzed. The recombinant protein interacts with calcyclin at a physiologically relevant range of Ca2+ in solution during affinity chromatography and on blots. Because CacyBP, like calcyclin, is present in the brain, the interaction of these two proteins might be involved in calcium signaling pathways in neuronal tissue.
Intermediate Filament Disassembly in Cultured Dorsal Root Ganglion Neurons Is Associated with Amino‐Terminal Head Domain Phosphorylation of Specific Subunits
Authors: Giasson Benoit I. , Mushynski Walter E.
Published: July 2010

Abstract: We previously reported that activation of protein kinase A in cultured rat dorsal root ganglion neurons, treated concomitantly with low concentrations of okadaic acid that selectively inhibit protein phosphatase‐2A, enhanced the Triton X‐100 solubility of neurofilament triplet proteins. We now show that peripherin and α‐internexin follow the same fragmentation profile as the neurofilament subunits, consistent with the notion that all five cytoplasmic intermediate filament proteins in these neurons form an integrated filamentous network whose assembly can be modulated by protein kinase A. Similar to the situation previously observed for the light neurofilament subunit, there was a strong correlation between phosphorylation of the amino‐terminal head domain of peripherin and filament fragmentation. In contrast, insignificant levels of 32P were incorporated into α‐internexin under conditions promoting disassembly, indicating that phosphorylation of this protein is not involved directly in filament fragmentation. The situation for the mid‐sized neurofilament subunit (NFM) was not as clear‐cut. Phosphopeptide mapping of NFM revealed many head and tail domain phosphorylation sites. However, changes in NFM head domain phosphorylation under conditions promoting filament disassembly were not as pronounced as for peripherin.
Activated Transcription of the Human Neuropeptide Y Gene in Differentiating SH‐SY5Y Neuroblastoma Cells Is Dependent on Transcription Factors AP‐1, AP‐2α, and NGFI
Authors: Wernersson Jonny , Johansson Irja , Larsson Ulrika , Minth‐Worby Carolyn , Påhlman Sven , Andersson Göran
Published: July 2010

Abstract: Activated transcription of the human neuropeptide Y gene (NPY) was investigated in SH‐SY5Y neuroblastoma cells at the onset of sympathetic neuronal differentiation induced by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) and serum or by nerve growth factor (NGF). As determined by transient expression, two NGF response elements (REs) were required for transcription induced by NGF in SH‐SY5Y cells with stable expression of an exogenous NGF receptor TRK‐A gene (SH‐SY5Y/trk). TPA treatment in the presence of serum induced NPY transcription in both wild‐type SH‐SY5Y (SH‐SY5Y/wt) and SH‐SY5Y/trk cells. A TPA RE (TRE), overlapping the proximal NGF RE, was identified by expression of the v‐Jun oncoprotein that enhanced NPY transcription. Suppression of TPA‐induced NPY transcription was obtained by expression of a dominant negative Jun protein, selective protein kinase C inhibition, or introduction of a mutated TRE, whereas NGF‐induced NPY transcription was inhibited to a lesser degree. The transcription factor AP‐2α was shown to bind cooperatively to the NPY promoter with either AP‐1 or NGFI‐A to the shared TRE and NGF RE and to the distal NGF RE, respectively. These results show that transcription factors AP‐1, AP‐2α, and NGFI‐A are involved in activated NPY transcription during the onset of neuronal differentiation.
NMDA Receptor Stimulation Selectively Initiates GABAA Receptor δ Subunit mRNA Expression in Cultured Rat Cerebellar Granule Neurons
Authors: Gault Laura M. , Siegel Ruth E.
Published: July 2010

Abstract: Findings in vivo and in culture suggest that neuronal activity selectively regulates GABAA receptor δ subunit mRNA expression in cerebellar granule neurons. For example, the onset of δ subunit mRNA expression during postnatal maturation coincides with innervation. Furthermore, depolarizing conditions (25 mM KCl) in culture initiate and maintain increases in the δ subunit transcript level. We have now examined whether similar changes in δ subunit mRNA expression occur in cultured neurons after activation of glutamate receptors of the NMDA subtype, an event that mimics granule neuron depolarization by mossy fiber innervation in vivo. Our studies demonstrate that addition of 50 µM NMDA to cultured rat granule neurons maintained in defined, serum‐free medium specifically initiates δ subunit transcript expression. Whereas the level of the δ subunit mRNA is increased fourfold by this treatment, levels of other GABAA receptor subunit transcripts are not significantly changed. The level of the δ subunit transcript is further increased when NMDA receptor activation is enhanced by maintaining neurons in a Mg2+‐free medium to alleviate Mg2+ blockade of the receptor channel. The NMDA‐induced elevation in δ subunit transcript expression involves activation of a Ca2+/calmodulin‐dependent protein kinase pathway. These findings suggest that activation of an excitatory pathway may regulate the expression of an inhibitory receptor phenotype in cerebellar granule neurons in vivo.
c‐Fos Deficiency Inhibits Induction of mRNA for Some, but Not All, Neurotransmitter Biosynthetic Enzymes by Immobilization Stress
Authors: Serova Lidia I. , Saez Enrique , Spiegelman Bruce M. , Sabban Esther L.
Published: July 2010

Abstract: Recent studies indicated that c‐Fos protein may be mediating stress‐elicited transcriptional activation of genes involved in neurotransmitter biosynthesis. However, direct evidence for c‐Fos mediating these changes in gene expression has been lacking. Mice with disrupted c‐fos gene (+/− or −/− genotypes) were used to examine the effect of immobilization stress on a group of stress‐responsive genes. In male adrenals, c‐Fos was found not essential for stress‐elicited activation of expression of tyrosine hydroxylase, dopamine β‐hydroxylase (DBH), phenylethanolamine N‐methyltransferase, or neuropeptide Y. In females, immobilization failed to induce adrenal DBH in the c‐Fos‐deficient mice. In brainstem, c‐Fos was indispensable for elevation of DBH mRNA in both genders. The gene, gender, and tissue specificity in the requirement for c‐Fos points to diversity in adaptation mechanisms to stress.
RGS7 Attenuates Signal Transduction Through the Gαq Family of Heterotrimeric G Proteins in Mammalian Cells
Authors: Shuey David J. , Betty Maria , Jones Philip G. , Khawaja Xavier Z. , Cockett Mark I.
Published: July 2010

Abstract: The RGS proteins are a recently discovered family of G protein regulators that have been shown to act as GTPase‐activating proteins (GAPs) on the Gαi and Gαq subfamilies of the heterotrimeric G proteins. Here, we demonstrate that RGS7 is a potent GAP in vitro on Gαi1 and Gαo heterotrimeric proteins and that RGS7 acts to down‐regulate Gαq‐mediated calcium mobilization in a whole‐cell assay system using a transient expression protocol. This RGS protein and RGS4 are reported to be expressed predominantly in brain, and in situ hybridization studies have revealed similarities in the regional distribution of RGS and Gαq mRNA expression. Our findings provide further evidence to support a functional role for RGS4 and RGS7 in Gαq‐mediated signaling in the CNS.
Increased MPTP Neurotoxicity in Vesicular Monoamine Transporter 2 Heterozygote Knockout Mice
Authors: Gainetdinov Raul R. , Fumagalli Fabio , Wang Yan‐Min , Jones Sara R. , Levey Allan I. , Miller Gary W. , Caron Marc G.
Published: July 2010

Abstract: The neurotoxic action of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) has been proposed to be attenuated by sequestration into intracellular vesicles by the vesicular monoamine transporter (VMAT2). The purpose of this study was to determine if mice with genetically reduced levels of VMAT2 (heterozygote knockout; VMAT2 +/−) were more vulnerable to MPTP. Striatal dopamine (DA) content, the levels of DA transporter (DAT) protein, and the expression of glial fibrillary acidic protein (GFAP) mRNA, a marker of gliosis, were assessed as markers of MPTP neurotoxicity. In all parameters measured VMAT2 +/− mice were more sensitive than their wild‐type littermates (VMAT2 +/+). Administration of MPTP (7.5, 15, or 30 mg/kg, b.i.d.) resulted in dose‐dependent reductions in striatal DA levels in both VMAT2 +/− and VMAT2 +/+ animals, but the neurotoxic potency of MPTP was approximately doubled in the VMAT2 +/− mice: 59 versus 23% DA loss 7 days after 7.5 mg/kg dose for VMAT2 +/− and VMAT2 +/+ mice, respectively. Dopaminergic nerve terminal integrity, as assessed by DAT protein expression, also revealed more drastic reductions in the VMAT2 +/− mice: 59 versus 35% loss at 7.5 mg/kg and 95 versus 58% loss at 15 mg/kg for VMAT2 +/− and VMAT2 +/+ mice, respectively. Expression of GFAP mRNA 2 days after MPTP was higher in the VMAT2 +/− mice than in the wild‐type: 15.8‐ versus 7.8‐fold increase at 7.5 mg/kg and 20.1‐ versus 9.6‐fold at 15 mg/kg for VMAT2 +/− and VMAT2 +/+ mice, respectively. These observations clearly demonstrate that VMAT2 +/− mice are more susceptible to the neurotoxic effects of MPTP, suggesting that VMAT2‐mediated sequestration of the neurotoxin into vesicles may play an important role in attenuating MPTP toxicity in vivo.
M1 Muscarinic Agonist Treatment Reverses Cognitive and Cholinergic Impairments of Apolipoprotein E‐Deficient Mice
Authors: Fisher A. , Brandeis R. , Chapman S. , Pittel Z. , Michaelson D. M.
Published: July 2010

Abstract: Recent studies suggest that apolipoprotein E (apoE) plays a specific role in brain cholinergic function and that the E4 allele of apoE (apoE4), a major risk factor for Alzheimer's disease (AD), may predict the extent of cholinergic dysfunction and the efficacy of cholinergic therapy in this disease. Animal model studies relevant to this hypothesis revealed that apoE‐deficient (knockout) mice have working memory impairments that are associated with distinct dysfunction of basal forebrain cholinergic neurons. Cholinergic replacement therapy utilizing M1‐selective muscarinic agonists has been proposed as effective treatment for AD patients. In the present study, we examined whether the memory deficits and brain cholinergic deficiency of apoE‐deficient mice can be ameliorated by the M1‐selective agonist 1‐methylpiperidine‐4‐spiro‐(2′‐methylthiazoline), [AF150(S)]. Treatment of apoE‐deficient mice with AF150(S) for 3 weeks completely abolished their working memory impairments. Furthermore, this reversal of cognitive deficit was associated with a parallel increase of histochemically determined brain choline acetyltransferase and acetylcholinesterase levels and with the recovery of these cholinergic markers back to control levels. These findings show that apoE deficiency‐related cognitive and cholinergic deficits can be ameliorated by M1‐selective muscarinic treatment. They also provide a novel model system for development and evaluation of therapeutic strategies directed specifically at the AD patients whose condition is attributed to the apoE genotype.
Oxindole, a Sedative Tryptophan Metabolite, Accumulates in Blood and Brain of Rats with Acute Hepatic Failure
Authors: Carpenedo Raffaella , Mannaioni Guido , Moroni Flavio
Published: July 2010

Abstract: Rats treated with oxindole (10–100 mg/kg i.p.), a putative tryptophan metabolite, showed decreased spontaneous locomotor activity, loss of the righting reflex, hypotension, and reversible coma. Brain oxindole levels were 0.05 ± 0.01 nmol/g in controls and increased to 8.1 ± 1.7 or 103 ± 15 nmol/g after its administration at doses of 10 or 100 mg/kg i.p., respectively. To study the role that oxindole plays in the neurological symptoms associated with acute liver failure, we measured the changes of its concentration in the brain after massive liver damage, and we investigated the possible metabolic pathways leading to its synthesis. Rats treated with either thioacetamide (0.2 and 0.4 g/kg i.p., twice) or galactosamine (1 and 2 g/kg i.p.) showed acute liver failure and a large increase in blood or brain oxindole concentrations (from 0.05 ± 0.01 nmol/g in brains of controls to 1.8 ± 0.3 nmol/g in brains of thioacetamide‐treated animals). Administration of tryptophan (300–1,000 mg/kg p.o.) caused a twofold increase, whereas administration of indole (10–100 mg/kg p.o.) caused a 200‐fold increase, of oxindole content in liver, blood, and brain, thus suggesting that indole formation from tryptophan is a limiting step in oxindole synthesis. Oral administration of neomycin, a broad‐spectrum, locally acting antibiotic agent able to reduce intestinal flora, significantly decreased brain oxindole content. Taken together, our data show that oxindole is a neurodepressant tryptophan metabolite and suggest that it may play a significant role in the neurological symptoms associated with acute liver impairment.
Regulation of Nicotinic Receptor Subtypes Following Chronic Nicotinic Agonist Exposure in M10 and SH‐SY5Y Neuroblastoma Cells
Authors: Warpman Ulrika , Friberg Linda , Gillespie Alison , Hellström‐Lindahl Ewa , Zhang Xiao , Nordberg Agneta
Published: July 2010

Abstract: The present study further investigated whether nicotinic acetylcholine receptor (nAChR) subtypes differ in their ability to up‐regulate following chronic exposure to nicotinic agonists. Seven nicotinic agonists were studied for their ability to influence the number of chick α4β2 nAChR binding sites stably transfected in fibroblasts (M10 cells) following 3 days of exposure. The result showed a positive correlation between the Ki values for binding inhibition and EC50 values for agonist‐induced α4β2 nAChR up‐regulation. The effects of epibatidine and nicotine were further investigated in human neuroblastoma SH‐SY5Y cells (expressing α3, α5, β2, and β4 nAChR subunits). Nicotine exhibited a 14 times lower affinity for the nAChRs in SH‐SY5Y cells as compared with M10 cells, whereas epibatidine showed similar affinities for the nAChRs expressed in the two cell lines. The nicotine‐induced up‐regulation of nAChR binding sites in SH‐SY5Y cells was shifted to the right by two orders of magnitude as compared with that in M10 cells. The epibatidine‐induced up‐regulation of nAChR binding sites in SH‐SY5Y cells was one‐fourth that in M10 cells. The levels of mRNA of the various nAChR subunits were measured following the nicotinic agonist exposure. In summary, the various nAChR subtypes show different properties in their response to chronic stimulation.
Identification of Cellular Compartments Involved in Processing of Cathepsin E in Primary Cultures of Rat Microglia
Authors: Sastradipura Dewi F. , Nakanishi Hiroshi , Tsukuba Takayuki , Nishishita Kazuhisa , Sakai Hideaki , Kato Yuzo , Gotow Takahiro , Uchiyama Yasuo , Yamamoto Kenji
Published: July 2010

Abstract: Cathepsin E is a major nonlysosomal, intracellular aspartic proteinase that localizes in various cellular compartments such as the plasma membrane, endosome‐like organelles, and the endoplasmic reticulum (ER). To learn the segregation mechanisms of cathepsin E into its appropriate cellular destinations, the present studies were initiated to define the biosynthesis, processing, and intracellular localization as well as the site of proteolytic maturation of the enzyme in primary cultures of rat brain microglia. Immunohistochemical and immunoblot analyses revealed that cathepsin E was the most abundant in microglia among various brain cell types, where the enzyme existed predominantly as the mature enzyme. Immunoelectron microscopy studies showed the presence of the enzyme predominantly in the endosome‐like vacuoles and partly in the vesicles located in the trans‐Golgi area and the lumen of ER. In the primary cultured microglial cells labeled with [35S]methionine, >95% of labeled cathepsin E were represented by a 46‐kDa polypeptide (reduced form) after a 30‐min pulse. Most of it was proteolytically processed via a 44‐kDa intermediate to a 42‐kDa mature form within 4 h of chase. This processing was completely inhibited by bafilomycin A1, a specific inhibitor of vacuolar‐type H+‐ATPase. Brefeldin A, a blocker for the traffic of secretory proteins from the ER to the Golgi complex, also inhibited the processing of procathepsin E and enhanced its degradation. Procathepsin E, after pulse‐labeling, showed complete susceptibility to endoglycosidase H, whereas the mature enzyme almost acquired resistance to endoglycosidases H as well as F. The present studies provide the first evidence that cathepsin E in microglia is first synthesized as the inactive precursor bearing high‐mannose oligosaccharides and processed to the active mature enzyme with complex‐type oligosaccharides via the intermediate form and that the final proteolytic maturation step occurs in endosome‐like acidic compartments.
Redistribution of Imipramine from Regions of the Brain Under the Influence of Circulating Specific Antibodies
Authors: Ragusi Corinne , Scherrmann Jean‐Michel , Harrison Kenneth , Smith David S. , Rips Richard , Boschi Gabrielle
Published: July 2010

Abstract: The kinetics of brain‐to‐blood redistribution of imipramine (IMI) was assessed in nine brain regions of control rats and rats given anti‐tricyclic antidepressant (anti‐TCA) antibody. Two antibodies were given intravenously 6 min after intravenous [3H]IMI (1 nmol/kg). One was a murine monoclonal IgG1 (Ka = 3.8 × 107M−1) at an IgG/IMI molar ratio of 1,000 (IgG1,000), and the other was a sheep polyclonal IgG (TAb; Ka = 1.3 × 1010M−1) at IgG/IMI molar ratios of 1, 10, and 100 (TAb1, TAb10, and TAb100). In the control rats, IMI was rapidly taken up by the brain (Cmax at 5 min) with no significant differences among the brain regions (4.1 ± 0.4 to 5.4 ± 0.6 pmol/g), and brain IMI then declined monoexponentially with a half‐life of 44.2 min (cerebellum) to 77.3 min (hippocampus). The greatest IMI content was in the frontal cortex and the lowest in the cerebellum. The antibodies (except TAb1) stimulated the extent and rate of IMI redistribution from all the brain regions depending on the immunoreactive capacity (NKa) of the antibody. The antibody with the highest NKa (TAb100) had the greatest effect. The fraction of IMI removed from the brain was 58–74%, and the redistribution half‐life was 7.9–15.6 min; the mean residence time was reduced by 66–75% (11.8–23.9 min). These results demonstrate that circulating anti‐TCA IgG rapidly and reliably removes IMI from the brain, indicating that immunotoxicotherapy could be an efficient procedure for accelerating the removal of TCA from the brain.
Modulation of the N‐Methyl‐d‐Aspartate Receptor by Haloperidol: NR2B‐Specific Interactions
Authors: Gallagher Michael J. , Huang Hui , Lynch David R.
Published: July 2010

Abstract: The dopaminergic antagonist haloperidol has an eight‐ to 10‐fold higher affinity for NMDA receptors containing the NR2B (ε2) subunit, showing the same subunit specificity as ifenprodil, polyamines, and magnesium. In the present study, we have compared the effects of mutations altering polyamine and ifenprodil sensitivity on haloperidol sensitivity of NMDA receptors. As seen for spermidine stimulation, high‐affinity haloperidol inhibition is governed by the region around amino acid 198, based on results from chimeric murine NR2A/NR2B (ε1/ε2) receptors. Mutation of ε2E201 in this region to asparagine or arginine causes a 10‐fold decrease in the ability of haloperidol to inhibit 125I‐MK‐801 binding. ε2E201 does not govern the interactions of ifenprodil, because all of the mutants at ε2E201 exhibited wild‐type affinity for ifenprodil. Mutation of ε2R337 causes a 400‐fold loss in apparent affinity for ifenprodil but does not change the effects of haloperidol. The structural determinants of spermidine stimulation do not perfectly match those for haloperidol inhibition, as mutations of E200 remove haloperidol inhibition but do not alter polyamine stimulation. The present results thus demonstrate that although spermidine, haloperidol, and ifenprodil share subunit selectivity and overlapping pharmacology, they also have specific structural determinants.
Histochemical Detection of Quinone Reductase Activity In Situ Using LY 83583 Reduction and Oxidation
Authors: Murphy Timothy H. , So Austin P. , Vincent Steve R.
Published: July 2010

Abstract: The application of enzymatic staining techniques, using tetrazolium dyes, to aldehyde‐treated brain sections has revealed the presence of NADPH‐diaphorase activity attributed to nitric oxide synthase. When evaluating the specificity of the putative guanylyl cyclase inhibitor LY 83583, a robust and novel staining pattern was noted in epithelial, endothelial, and astrocytic cells when LY 83583 was included in the NADPH‐diaphorase histochemical reaction. This LY 83583‐dependent staining could be blocked by the NAD(P)H:quinone oxidoreductase inhibitor dicumarol. Based on its quinone structure, we hypothesized that LY 83583 was a substrate for the enzyme NAD(P)H:quinone oxidoreductase. Transfection of human embryonic kidney 293 cells with the rat liver isoform of NAD(P)H:quinone oxidoreductase resulted in robust NADPH‐ and LY 83583‐dependent staining that was completely blocked by dicumarol and was not observed in untransfected cells. Analysis of transfected cell extracts and brain homogenates indicated that LY 83583 was a substrate for NAD(P)H:quinone oxidoreductase, with a Km similar to the well‐characterized substrate menadione. Sensitivity of the nitroblue tetrazolium reduction to superoxide dismutase indicated that the reduction of LY 83583 by NAD(P)H:quinone oxidoreductase leads to superoxide generation. The localization of NAD(P)H:quinone oxidoreductase activity to astrocytic cells suggests a role for glia in combating oxidative insults to brain and in activating quinone‐like drugs such as LY 83583.
Artefacts in HPLC Detection of 3‐Nitrotyrosine in Human Brain Tissue
Authors: Kaur Harparkash , Lyras Leonidas , Jenner Peter , Halliwell Barry
Published: July 2010

Abstract: An HPLC method was used for quantification of 3‐nitrotyrosine (3‐NT) in human postmortem brain tissue. A peak with similar retention time to 3‐NT was detected in brain tissue from patients with Parkinson's disease, Huntington's chorea, multiple system atrophy, and Alzheimer's disease but not in control tissue. The peak was lost on reduction with dithionite, a criterion often used to identify 3‐NT. Tissue from the same neurodegenerative diseases was analysed by HPLC using a photodiode array detector in series with an amperometric electrochemical detector, but the peak was found not to be 3‐NT. The absorbance spectrum, fragmentation pattern on mass spectroscopy, and electrochemical profile of this peak do not match authentic 3‐NT. A search of the mass spectroscopy databases failed to reveal its identity. The presence of this closely eluting, dithionite‐reducible peak could confound analysis of human tissues for 3‐NT. In vitro experiments showed that high concentrations of peroxynitrite were needed to achieve detectable levels of 3‐NT in human brain tissue.
Effects of C‐Terminal Truncation of the Recombinant δ‐Opioid Receptor on Phospholipase C and Adenylyl Cyclase Coupling
Authors: Hirst Robert A. , Smart Darren , Devi Lakshmi A. , Lambert David G.
Published: July 2010

Abstract: Opioid receptors belong to the superfamily of guanine nucleotide binding (G) protein‐coupled receptors. There is now growing evidence in support of a stimulatory coupling of opioid receptors to phospholipase C (PLC), via a pertussis toxin‐sensitive G protein, leading to the generation of the second messenger inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3]. We have generated two C‐terminal truncation mutants of the δ‐opioid receptor lacking the final 15 or 37 amino acids and examined their coupling to PLC and adenylyl cyclase. d‐[Pen2,5]‐enkephalin (DPDPE) mediated Ins(1,4,5)P3 formation and cyclic AMP inhibition was measured in whole cells and assayed using radioreceptor mass assays. DPDPE produced a time‐ and dose‐dependent increase in Ins(1,4,5)P3 mass formation in Chinese hamster ovary (CHO) cells expressing the δwt, δ15, and δ37 receptors. As the C terminus was truncated, the time to maximum stimulation (15 s in CHOδwt, 60 s in CHOδ15, and 120 s in CHOδ37) increased and removal of the C terminus resulted in a prompt return to basal Ins(1,4,5)P3 levels. Whereas the dose‐response curves to Ins(1,4,5)P3 formation and cyclic AMP inhibition remained largely unaffected by C‐terminal truncation, there were large differences in the pEC/IC50 values, with cyclic AMP inhibition being the more potent, perhaps indicating Giα coupling to adenylyl cyclase and Giβ/γ coupling to PLC. Collectively, these data indicate that the C terminus of the δ‐opioid receptor is unimportant in the acute coupling to adenylyl cyclase but may have a role to play in PLC coupling. We hypothesize that an intact C terminus is required to allow normal “strong” coupling of receptor to Gi and that truncation weakens this link as reflected in an increased time to peak. In addition, if the coupling is weak, the acute response to agonist stimulation rapidly uncouples.
Intracellular Free Calcium Dynamics in Stretch‐Injured Astrocytes
Authors: Rzigalinski B. A. , Weber J. T. , Willoughby K. A. , Ellis E. F.
Published: July 2010

Abstract: We have previously developed an in vitro model for traumatic brain injury that simulates a major component of in vivo trauma, that being tissue strain or stretch. We have validated our model by demonstrating that it produces many of the posttraumatic responses observed in vivo. Sustained elevation of the intracellular free calcium concentration ([Ca2+]i) has been hypothesized to be a primary biochemical mechanism inducing cell dysfunction after trauma. In the present report, we have examined this hypothesis in astrocytes using our in vitro injury model and fura‐2 microphotometry. Our results indicate that astrocyte [Ca2+]i is rapidly elevated after stretch injury, the magnitude of which is proportional to the degree of injury. However, the injury‐induced [Ca2+]i elevation is not sustained and returns to near‐basal levels by 15 min postinjury and to basal levels between 3 and 24 h after injury. Although basal [Ca2+]i returns to normal after injury, we have identified persistent injury‐induced alterations in calcium‐mediated signal transduction pathways. We report here, for the first time, that traumatic stretch injury causes release of calcium from inositol trisphosphate‐sensitive intracellular calcium stores and may uncouple the stores from participation in metabotropic glutamate receptor‐mediated signal transduction events. We found that for a prolonged period after trauma astrocytes no longer respond to thapsigargin, glutamate, or the inositol trisphosphate‐linked metabotropic glutamate receptor agonist trans‐(1S,3R)‐1‐amino‐1,3‐cyclopentanedicarboxylic acid with an elevation in [Ca2+]i. We hypothesize that changes in calcium‐mediated signaling pathways, rather than an absolute elevation in [Ca2+]i, is responsible for some of the pathological consequences of traumatic brain injury.
SCLIP: A Novel SCG10‐Like Protein of the Stathmin Family Expressed in the Nervous System
Authors: Ozon Sylvie , Byk Tamara , Sobel André
Published: July 2010

Abstract: Stathmin is a cytosolic phosphoprotein previously described as a ubiquitous relay integrating various signaling pathways. It is the generic element of a protein family in mammals as in Xenopus, including SCG10, a neuron‐specific, growth‐associated protein, and RB3/XB3, related to the expression of differentiated functions of mature cells of the nervous system. In an extensive search for other members of the stathmin family, we identified cDNAs coding for two novel stathmin‐related proteins: (a) a cDNA from a rat striatum cDNA library codes for RB3″, a splice variant of RB3, with an additional basic domain in its N‐terminal region; and (b) another cDNA identified through a systematic search in EST databases codes for a novel protein, SCLIP, for “SCG10‐like protein,” displaying 70% identity with SCG10 and sharing the same domain organization, with an N‐terminal domain likely involved in membrane attachment and a C‐terminal stathmin‐like domain. Northern blot analysis as well as in situ hybridization on 14‐day rat embryos showed that SCLIP mRNA is expressed only in neural structures. SCLIP mRNA is expressed at comparable levels in neonatal and adult rat brain, suggesting a potential role not only in the acquisition, but also in the expression of differentiated neuronal functions.
Cyclic AMP‐Elevating Agents Prevent Oligodendroglial Excitotoxicity
Authors: Yoshioka Akira , Shimizu Yuko , Hirose Genjiro , Kitasato Hiroshi , Pleasure David
Published: July 2010

Abstract: Previously, we have demonstrated that cells of the oligodendroglial lineage express non‐NMDA glutamate receptor genes and are damaged by kainate‐induced Ca2+ influx via non‐NMDA glutamate receptor channels, representing oligodendroglial excitotoxicity. We find in the present study that agents that elevate intracellular cyclic AMP prevent oligodendroglial excitotoxicity. After oligodendrocyte‐like cells, differentiated from the CG‐4 cell line established from rat oligodendrocyte type‐2 astrocyte progenitor cells, were exposed to 2 mM kainate for 24 h, cell death was evaluated by measuring activity of lactate dehydrogenase released into the culture medium. Released lactate dehydrogenase increased about threefold when exposed to 2 mM kainate. Kainate‐induced cell death was prevented by one of the following agents: adenylate cyclase activator (forskolin), cyclic AMP analogues (dibutyryl cyclic AMP and 8‐bromo‐cyclic AMP), and cyclic AMP phosphodiesterase inhibitors (3‐isobutyl‐1‐methylxanthine, pentoxifylline, propentofylline, and ibudilast). Simultaneous addition of both forskolin and phosphodiesterase inhibitors prevented the kainate‐induced cell death in an additive manner. A remarkable increase in Ca2+ influx (∼5.5‐fold) also was induced by kainate. The cyclic AMP‐elevating agents caused a partial suppression of the kainate‐induced increase in Ca2+ influx, leading to a less prominent response of intracellular Ca2+ concentration to kainate. The suppressing effect of forskolin on the kainate‐induced Ca2+ influx was partially reversed by H‐89, an inhibitor of cyclic AMP‐dependent protein kinase. In contrast to this, okadaic acid, an inhibitor of protein phosphatases 1 and 2A, brought about a decrease in the kainate‐induced Ca2+ influx. We therefore concluded that cyclic AMP‐elevating agents prevented oligodendroglial excitotoxicity by cyclic AMP‐dependent protein kinase‐dependent protein phosphorylation, resulting in decreased kainate‐induced Ca2+ influx.
CCAAT/Enhancer Binding Protein β Is a Neuronal Transcriptional Regulator Activated by Nerve Growth Factor Receptor Signaling
Authors: Sterneck Esta , Johnson Peter F.
Published: July 2010

Abstract: CCAAT/enhancer binding protein β (C/EBPβ) is a transcriptional regulator of the basic leucine zipper family. By in situ hybridization analysis, we found that C/EBPβ is widely expressed in the CNS of adult mice, including cells of the hippocampus and dentate gyrus and cerebellar Purkinje and granule cells. Expression of C/EBPβ had also been reported in the PC12 cell line, which undergoes differentiation to neuron‐like cells in response to nerve growth factor (NGF). We show that C/EBPβ mRNA expression increases while protein levels decrease during differentiation of PC12 cells. In transactivation assays, C/EBPβ activity was stimulated by NGF receptor signaling. Mutations of a phosphorylation site for mitogen‐activated protein kinase in C/EBPβ affected its capacity to transactivate in a promoter‐specific manner. Our data identify the C/EBPβ protein and gene as direct downstream targets of the NGF receptor and suggest a role for C/EBPβ in neurotrophin signaling in the brain.
In Vitro Studies of Ferritin Iron Release and Neurotoxicity
Authors: Double K. L. , Maywald M. , Schmittel M. , Riederer P. , Gerlach M.
Published: July 2010

Abstract: The increase in brain iron associated with several neurodegenerative diseases may lead to an increased production of free radicals via the Fenton reaction. Intracellular iron is usually tightly regulated, being bound by ferritin in an insoluble ferrihydrite core. The neurotoxin 6‐hydroxydopamine (6‐OHDA) releases iron from the ferritin core by reducing it to the ferrous form. Iron release induced by 6‐OHDA and structurally related compounds and two other dopaminergic neurotoxins, 1‐methyl‐4‐phenylpyridinium iodide (MPP+) and 1‐trichloromethyl‐1,2,3,4‐tetrahydro‐β‐carboline (TaClo), were compared, to identify the structural characteristics important for such release. 1,2,4‐Trihydroxybenzene (THB) was most effective in releasing ferritin‐bound iron, followed by 6‐OHDA, dopamine, catechol, and hydroquinone. Resorcinol, MPP+, and TaClo were ineffective. The ability to release iron was associated with a low oxidation potential. It is proposed that a low oxidation potential and an ortho‐dihydroxyphenyl structure are important in the mechanism by which ferritin iron is mobilized. In the presence of ferritin, both 6‐OHDA and THB strongly stimulated lipid peroxidation, an effect abolished by the addition of the iron chelator deferoxamine. These results suggest that ferritin iron release contributes to free radical‐induced cell damage in vivo.
Optical Imaging of Hippocampal Neurons with a Chloride‐Sensitive Dye: Early Effects of In Vitro Ischemia
Authors: Inglefield Jon R. , Schwartz‐Bloom Rochelle D.
Published: July 2010

Abstract: We determined if changes in intraneuronal Cl− occur early after ischemia in the hippocampal slice. Slices from juvenile rats (14–19 days old) were loaded with the cell‐permeant form of 6‐methoxy‐N‐ethylquinolinium chloride (MEQ), a Cl−‐sensitive fluorescent dye. Real‐time changes in intracellular chloride concentration ([Cl−]i) were measured with UV laser scanning confocal microscopy in multiple neurons within each slice. In vitro ischemia (26–28°C, 10 min) was confirmed by the loss of synaptic transmission (evoked field excitatory postsynaptic potentials) from pyramidal cells in area CA1. After ischemia and reoxygenation (10 min), MEQ fluorescence decreased significantly in CA1 pyramidal cells and interneurons. The decreased fluorescence corresponded to an ischemia‐induced increase in [Cl−]i of ∼10 mM. Pretreatment with the GABAA‐gated Cl− channel antagonist picrotoxin (100 µM) blocked the ischemia‐induced change in [Cl−]i. Analysis of the superfusates indicated that ischemia also caused a transient amino acid (GABA, glutamate, and aspartate) release that was maximal at ∼10 min, returning to baseline shortly thereafter. Recovery from ischemia was confirmed by the return of synaptic transmission in area CA1, the return toward baseline of the ischemia‐induced decrease in MEQ fluorescence, and exclusion of propidium iodide from MEQ fluorescent cells. Furthermore, pyramidal cells did not undergo cell swelling during this early phase of reoxygenation, as indicated by the volume‐sensitive dye calcein. Thus, mild ischemia induces the accumulation of [Cl−]i secondary to GABAA receptor activation, in the absence of cellular swelling or death. In contrast, depolarization of the slice with K+ (50 mM) decreased MEQ fluorescence significantly but caused cell swelling. Picrotoxin did not prevent the K+‐induced increase in [Cl−]i. It is possible that an increased [Cl−]i, following either an ischemic event or an episode of depolarization, would reduce the Cl− driving force and thereby limit synaptic transmission by GABA. To support this hypothesis, ischemia caused a reduction in the ability of the GABA agonist muscimol to increase [Cl−]i after 20‐min reoxygenation.
Membrane Instability, Plasmalogen Content, and Alzheimer's Disease
Authors: Ginsberg Lionel , Xuereb John H. , Gershfeld Norman L.
Published: July 2010

Abstract: The normal stability of the cell membrane bilayer depends on its lipid composition being appropriate to the ambient (physiological) temperature, Tp. Membrane lipid composition may be altered by disease such that the bilayer is only stable at a new critical temperature, T⋆, which may differ from Tp. In Alzheimer's disease (AD) temporal cortex, a defect of lipid composition has previously been identified, namely, a decrease in the ratio of plasmalogen to nonplasmalogen ethanolamine glycerophospholipids. Furthermore, for AD temporal cortex neural membranes, T⋆≪ Tp, a finding confirmed in the present study in a larger series than previously, using a new method for obtaining T⋆. This inequality between T⋆ and Tp has been proposed as a putative contributory pathogenetic mechanism leading to membrane destabilisation in AD brain. The plasmalogen deficiency could account for the change in T⋆ in AD, as shown by experiments where T⋆ was measured for artificial lipid mixtures simulating brain membranes with varying plasmalogen/nonplasmalogen ratios. The critical temperature was found to be very sensitive to small alterations in plasmalogen content.
Distinct Effects of Imipramine on 5‐Hydroxytryptamine Uptake Mediated by the Recombinant Rat Serotonin Transporter SERT1
Authors: Sur Cyrille , Betz Heinrich , Schloss Patrick
Published: July 2010

Abstract: Tricyclic and nontricyclic serotonin [5‐hydroxytryptamine (5‐HT)] uptake inhibitors are widely used for the treatment of depression. Here, we show that both the tricyclic antidepressant imipramine and the nontricyclic antidepressant citalopram competitively inhibit 5‐HT transport mediated by the recombinant rat 5‐HT transporter SERT1. For citalopram, the concentration producing half‐maximal transport inhibition was in the same order of magnitude as its KD value determined by equilibrium binding. In contrast, the inhibitory potency of imipramine was more than one order of magnitude lower than its KD value. Our data are consistent with low‐affinity imipramine binding occurring at or close to the substrate recognition site, which also binds citalopram. Occupation of the high‐affinity imipramine binding site on SERT1 did not affect 5‐HT transport but allosterically displaced citalopram from the substrate recognition site. Consequently, low concentrations of imipramine partially protected 5‐HT transport from citalopram inhibition. This protection was only observed in the presence of Na+ because high‐affinity imipramine binding is strictly sodium‐dependent. Thus, depending on which of its binding sites on SERT1 is occupied, imipramine may exert distinct effects on 5‐HT uptake mediated by the recombinant rat 5‐HT transporter.
Calmodulin Modulates Mitogen‐Activated Protein Kinase Activation in Response to Membrane Depolarization in PC12 Cells
Authors: Egea Joaquim , Espinet Carme , Comella Joan X.
Published: July 2010

Abstract: In the absence of neurotrophic factors, chronic depolarization of plasma membrane has been shown to maintain several populations of primary neurons in culture. We report that in the PC12 cell line, depolarization causes Ca2+ influx through voltage‐gated Ca2+ channels, which is able to stimulate extracellular‐regulated kinase (ERK) activity. We studied which mediators were responsible for ERK activation resulting from increased levels of Ca2+ in the cytoplasm and found that calmodulin was involved in this process. The addition of W13, a calmodulin inhibitor, to the culture medium, prevented ERK activation when PC12 cells were depolarized. In addition, we show that high K+ treatment did not induce Trk A phosphorylation, thus excluding the possibility of Ca2+ operating through this receptor to activate the ERK signal transduction pathway. Moreover, although high K+ treatment is able to phosphorylate the epidermal growth factor receptor (EGFR) and thus to activate the ERK signal transduction pathway, we demonstrate that W13 did not alter the state of EGFR phosphorylation in conditions that almost completely blocked ERK activation. These data suggest that calmodulin mediates ERK activation induced by increases in intracellular Ca2+ concentration in PC12 cells by a mechanism that seems to be independent of Trk A and EGFR activation.
Identification of Lectin‐Purified Neural Glycoproteins, GPs 180, 116, and 110, with NMDA and AMPA Receptor Subunits: Conservation of Glycosylation at the Synapse
Authors: Clark R. A. C. , Gurd J. W. , Bissoon N. , Tricaud N. , Molnar E. , Zamze S. E. , Dwek R. A. , McIlhinney R. A. J. , Wing D. R.
Published: July 2010

Abstract: The postsynaptic apparatus is associated with a number of glycoproteins with apparent molecular masses of 180, 116, and 110 kDa, which are highly concentrated in and may be uniquely associated with this structure. These glycoproteins, purified by concanavalin A lectin‐affinity chromatography, showed immunoreactivity in the present study with subunit‐specific antibodies to glutamate receptors as follows: GP 180, NMDA receptor subunits NR2A/NR2B; GP 116, NMDA receptor NR1 (1a); and GP 110, pan‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (pan‐AMPA) receptors. Sensitivities to the glycosidases peptide N‐glycosidase F and endo‐β‐N‐acetylglucosaminidase H on both western blots and silver‐stained gels suggested that the glutamate receptors were at least major constituents of the glycoprotein bands. Similar detailed glycosylation was observed for all three glycoproteins, with neutral oligosaccharides being dominant. Oligomannosidic glycans (with from five to nine mannoses) accounted for ∼50% of the neutral sugars, with Man 5 (at almost 20% of the neutral sugars) always the major glycan. Other abundant neutral oligosaccharides were of the complex type. Similar sensitivities to peptide N‐glycosidase F and endo‐β‐N‐acetylglucosaminidase H were observed for cell line‐expressed NMDA receptor subunits, suggesting that irrespective of the glycosylation processing available, the least highly processed oligosaccharides will be expressed. This may be indicative of glycosylation sites in these receptors that are inaccessible to the later processing enzymes and favours the oligomannosidic class of glycans in functional roles.
Caspases Mediate 6‐Hydroxydopamine‐Induced Apoptosis but Not Necrosis in PC12 Cells
Authors: Ochu Erinma E. , Rothwell Nancy J. , Waters Catherine M.
Published: July 2010

Abstract: The neurotoxin 6‐hydroxydopamine (6‐OHDA) induces apoptosis in the rat phaeochromocytoma cell line PC12. 6‐OHDA‐induced apoptosis is morphologically indistinguishable from serum deprivation‐induced apoptosis. Exposure of PC12 cells to a low concentration of 6‐OHDA (25 µM) results in apoptosis, whereas an increased concentration (50 µM) results in a mixture of apoptosis and necrosis. We investigated the involvement of caspases in the apoptotic death of PC12 cells induced by 6‐OHDA, using a general caspase inhibitor, benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethyl ketone (zVAD‐fmk), and compared this with serum deprivation‐induced apoptosis, which is known to involve caspases. We show that zVAD‐fmk (100 µM) completely prevented the apoptotic morphology of chromatin condensation induced by exposure to either 6‐OHDA (25 and 50 µM) or serum deprivation. Furthermore, cell lysates from 6‐OHDA‐treated cultures showed cleavage of a fluorogenic substrate for caspase‐3‐like proteases (caspase‐2, 3, and 7), acetyl‐Asp‐Glu‐Val‐Asp‐aminomethylcoumarin, and this was inhibited by zVAD‐fmk. However, although zVAD‐fmk restored total cell viability to serum‐deprived cells or cells exposed to 25 µM 6‐OHDA, the inhibitor did not restore viability to cells exposed to 50 µM 6‐OHDA. These data show the involvement of a caspase‐3‐like protease in 6‐OHDA‐induced apoptosis and that caspase inhibition is sufficient to rescue PC12 cells from the apoptotic but not the necrotic component of 6‐OHDA neurotoxicity.
Cytokine‐Induced Accumulation of Very Long‐Chain Fatty Acids in Rat C6 Glial Cells: Implication for X‐Adrenoleukodystrophy
Authors: Khan Mushfiquddin , Pahan Kalipada , Singh Avtar K. , Singh Inderjit
Published: July 2010

Abstract: X‐Adrenoleukodystrophy (X‐ALD) is an inherited metabolic disorder of very long‐chain fatty acids (VLCFA) with subsequent manifestation of neuroinflammatory disease. To investigate the possible role of proinflammatory cytokines in the X‐ALD disease process, we examined the effect of cytokines on the metabolism of VLCFA in C6 glial cells expressing oligodendrocyte‐like properties. C6 glial cells under serum‐free conditions were treated with different combinations of cytokines (tumor necrosis factor‐α, interleukin‐1β, interferon‐γ) or cytokine with bacterial lipopolysaccharide (LPS). Cytokine‐treated C6 cells had higher concentrations of VLCFA, measured as percent weight and also as C26:0/C22:0 ratio, which were 300–400% as compared with the controls. We also found increased levels of C26:1 in cytokine‐treated cells. The accumulation of VLCFA paralleled the decrease (35–55%) in peroxisomal β‐oxidation activity and a 12‐ to 14‐fold increase in the production of nitric oxide (NO). Individual cytokines were unable either to produce NO or to increase the levels of VLCFA in C6 cells. Inhibition of cytokine‐induced NO production by l‐N‐methylarginine, an inhibitor of NO synthase (NOS), and N‐acetylcysteine, an inhibitor of cytokine‐mediated induction of inducible NOS, normalized the peroxisomal β‐oxidation activity and the levels of VLCFA, suggesting a role for the proinflammatory cytokines and NO toxicity in the neuropathological changes associated with abnormal VLCFA metabolism (e.g., X‐ALD). X‐ALD is a peroxisomal disease having deficient oxidation of VLCFA, resulting in the excessive accumulation of VLCFA in all tissues but especially in brain. We observed greater increase in levels of VLCFA in the inflammatory region of ALD brain (in the demyelinating plaque and the area around the plaque) than in the normal‐looking area away from the plaque; this also indicates that cytokines in the proinflammatory region may augment the VLCFA defect caused by the inherited abnormality in X‐ALD brain. Although C6 glial cultured cells do not reflect the X‐ALD model precisely, the observed relationship between the cytokine‐induced inhibition of the oxidation of VLCFA, excessive accumulation of VLCFA, and excessive production of NO and their normalization by inhibitors of NOS in C6 glial cells suggests that NO‐mediated toxicity may play a role in VLCFA‐associated neuroinflammatory diseases (e.g., X‐ALD).