bluuebunny/crossref_metadata_2025 huggingface dataset:
Nanomolar Amyloid β Protein‐Induced Inhibition of Cellular Redox Activity in Cultured Astrocytes
Authors: Kato Mayumi , Saito Hiroshi , Abe Kazuho
Published: July 2010

Abstract: It has been previously reported that Alzheimer's amyloid β protein (Aβ) induces reactive astrocytosis in culture. In the present study, we found that Aβ potently inhibits cellular redox activity of cultured astrocytes, as determined by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide reduction assay. The following comparative studies revealed several differences between these two actions of Aβ on astrocytes. First, Aβ‐induced reactive morphological change was suppressed by the presence of serum or thrombin, and Aβ inhibition of cellular redox activity was observed in either the presence or the absence of serum. Second, micromolar concentrations (10 µM or more) were required for Aβ to induce reactive astrocytosis, whereas nanomolar concentrations (0.1–100 nM) were sufficient to inhibit cellular redox activity. Third, the effect of micromolar Aβ was virtually irreversible, but nanomolar Aβ‐induced inhibition of cellular redox activity was reversed by washing out Aβ. Furthermore, as it has been reported that Aβ neurotoxicity is mediated by reactive oxygen species, we also examined if similar mechanisms are involved in astrocytic response to Aβ. However, neither Aβ‐induced morphological change nor inhibition of redox activity was blocked by antioxidants, suggesting that these effects are not caused by oxidative stress.
Intracellular Concentrations of Major Ions in Rat Myelinated Axons and Glia: Calculations Based on Electron Probe X‐Ray Microanalyses
Authors: Stys Peter K. , Lehning Ellen , Saubermann A. J. , LoPachin Richard M.
Published: July 2010

Abstract: Electron probe x‐ray microanalysis (EPMA) was used to measure water content (percent water) and dry weight elemental concentrations (in millimoles per kilogram) of Na, K, Cl, and Ca in axoplasm and mitochondria of rat optic and tibial nerve myelinated axons. Myelin and cytoplasm of glial cells were also analyzed. Each anatomical compartment exhibited characteristic water contents and distributions of dry weight elements, which were used to calculate respective ionized concentrations. Free axoplasmic [K+] ranged from ≈155 mM in large PNS and CNS axons to ≈120–130 mM in smaller fibers. Free [Na+] was ≈15–17 mM in larger fibers compared with 20–25 mM in smaller axons, whereas free [Cl−] was found to be 30–55 mM in all axons. Because intracellular Ca is largely bound, ionized concentrations were not estimated. However, calculations of total (free plus bound) aqueous concentrations of this element showed that axoplasm of large CNS and PNS axons contained ≈0.7 mM Ca, whereas small fibers contained 0.1–0.2 mM. Calculated ionic equilibrium potentials were as follows (in mV): in large CNS and PNS axons, EK = −105, ENa = 60, and ECl = −28; in Schwann cells, EK = −107, ENa = 33, and ECl = −33; and in CNS glia, EK = −99, ENa = 36, and ECl = −44. Calculated resting membrane potentials were as follows (in mV, including the contribution of the Na+,K+‐ATPase): large axons, about −80; small axons, about −72 to −78; and CNS glia, −91. ECl is more positive than resting membrane potential in PNS and CNS axons and glia, indicating active accumulation. Direct EPMA measurement of elemental concentrations and subsequent calculation of ionized fractions in axons and glia offer fundamental neurophysiological information that has been previously unattainable.
Zinc Allosterically Modulates Antagonist Binding to Cloned D1 and D2 Dopamine Receptors
Authors: Schetz John A. , Sibley David R.
Published: July 2010

Abstract: Cations of various size and charge were used as atomic scale probes of D1 and D2 dopamine receptors. Those cations that perturbed the binding of D1‐ and D2‐selective dopamine receptor antagonists were identified by screening at 5 mM cation. Pseudo‐noble‐gas‐configuration d‐transition metals, such as zinc, exerted a complete inhibition of specific binding, whereas most other cations had little or no effect. The nature of zinc's actions was characterized by measuring the radioligand binding properties of [3H]SCH‐23390 and [3H]methylspiperone to cloned D1A and D2L dopamine receptors in either the presence or absence of Zn2+. Zinc exerts a low‐affinity, dose‐dependent, EDTA‐reversible inhibition of the binding of subtype‐specific antagonists primarily by decreasing the ligands' affinity for their receptors. The mechanism of zinc inhibition appears to be allosteric modulation of the dopamine receptor proteins because zinc increases the dissociation constant (KD) of ligand binding, Schild‐type plots of zinc inhibition reach a plateau, and zinc accelerates antagonist dissociation rates. Here we demonstrate the effect of zinc on the binding of D1‐ and D2‐selective antagonists to cloned dopamine receptors and show that the inhibition by zinc is through a dose‐dependent, reversible, allosteric, two‐state modulation of dopamine receptors.
Corticotropin‐Releasing Factor (CRF), CRF‐Binding Protein (CRF‐BP), and CRF/CRF‐BP Complex in Alzheimer's Disease and Control Postmortem Human Brain
Authors: Behan Dominic P. , Khongsaly On , Owens Michael J. , Chung Hyung D. , Nemeroff Charles B. , De Souza Errol B.
Published: July 2010

Abstract: In Alzheimer's disease (AD) there are dramatic reductions in human corticotropin‐releasing factor (hCRF) concentration and reciprocal increases in CRF receptor density in the cortex. hCRF‐binding protein (hCRF‐BP), hCRF/hCRF‐BP complex, and “free” hCRF were measured in 10 brain regions from control and AD postmortem human tissue. In the control brains hCRF‐BP was heterogenously distributed and levels were at least 10‐fold higher on a molar basis than total hCRF levels, suggesting that one major role of the binding protein is to limit the actions of hCRF at the hCRF receptors. Concordant with this hypothesis, the percentage of total hCRF that was in the bound inactive form ranged from 65 to 90% in most areas examined, with the exception of the caudate and globus pallidus where only 15 and 40% were complexed, respectively. hCRF‐BP concentrations were similar in the control and AD groups except for Brodmann area (BA) 39 where there was a small but significant decrease in the AD group. Complexed hCRF levels were significantly decreased in BA 8/BA 9, BA 22, BA 39, nucleus basalis, and globus pallidus in the Alzheimer's group and free hCRF levels were significantly decreased only in three brain areas, BA 4, BA 39, and caudate; substantial (40%) but nonsignificant decreases were also noted in BA 8/BA 9 and BA 22. These data demonstrate that (1) a large proportion of the total hCRF in human brain is complexed to hCRF‐BP and thus unavailable for hCRF receptor activation, (2) reductions in total hCRF alone do not necessarily predict reductions in bioactive free hCRF, and (3) total hCRF levels and hCRF‐BP levels appear to be the main factors determining the quantity of bound and free hCRF in human brain.
Unusual Gangliosidosis in Emu (Dromaius novaehollandiae)
Authors: Freischütz Bettina , Tokuda Akira , Ariga Toshio , Bermudez Alex J. , Yu Robert K.
Published: July 2010

Abstract: A previous study has demonstrated an unusual gangliosidosis in emu that is characterized by the accumulation of gangliosides in the brain tissues with GM3 and GM1 predominating. To provide insight into this unique disorder of emu gangliosidosis, the current study focused on analysis of neutral glycosphingolipids and gangliosides from brain and liver tissues of affected birds and healthy controls. We found not only that the total lipid‐bound sialic acid content was increased three‐ and fourfold in the affected brain and liver, respectively, but also that the ganglioside pattern was rather complex as compared with the control. The absolute ganglioside sialic acid content was significantly increased in the diseased tissues, with the highest elevation levels of GM3 (14‐fold) and GM1 (ninefold) in the affected brain. Relative increases in content of these monosialogangliosides were also significant. GM2 was only detected in the affected brain, but not in normal controls. The neutral glycosphingolipid fraction showed accumulation of many oligosylceramides, with six‐ and 5.5‐fold increases in lactosylceramide levels for brain and liver, respectively. The level of myelin‐associated galactosylceramide (GalCer) in the brain was decreased to only 41% of that in the healthy control, whereas no difference was found in liver tissues from both groups. Besides GalCer, the brain content of sulfatide (cerebroside‐sulfate esters), another myelin‐associated glycolipid, decreased to only 16% of the control. The loss of myelin‐associated GalCer and sulfatide strongly suggests demyelination in the affected emu brain. Our overall data are consistent with the presence of a unique form of sphingolipidosis in the affected emus, perhaps with secondary demyelination, and suggest a metabolic disorder related to total sphingolipid activator deficiency.
Glycosaminoglycan Storage in Cultured Neonatal Murine Mucopolysaccharidosis Type VII Neuroglial Cells and Correction by β‐Glucuronidase Gene Transfer
Authors: Taylor Rosanne M. , Wolfe John H.
Published: July 2010

Abstract: The inherited deficiency of β‐glucuronidase activity causes the lysosomal storage disorder mucopolysaccharidosis (MPS) type VII (Sly disease). The sequential catabolism of glycosaminoglycans in lysosomes is blocked, and undegraded substrates accumulate in cells of many tissues, including neurons and glia in the brain. To evaluate the deficient metabolic pathway, primary cultures of mixed brain cells were established from newborn MPS VII mice. β‐Glucuronidase levels and glycosaminoglycan accumulation were studied in normal, carrier, and MPS VII cells. Retroviral vector‐mediated transfer of a normal β‐glucuronidase cDNA corrected the enzymatic deficiency in MPS VII cells and restored glycosaminoglycan catabolism to normal. High levels of β‐glucuronidase expression were sustained in vector‐corrected nondividing glial cell cultures for >2 months. These studies provide an in vitro model for evaluating somatic gene transfer in neural cells affected in mucopolysaccharidoses.
Differential Inactivation of Postsynaptic Density‐Associated and Soluble Ca2+/Calmodulin‐Dependent Protein Kinase II by Protein Phosphatases 1 and 2A
Authors: Strack Stefan , Barban Mary Ann , Wadzinski Brian E. , Colbran Roger J.
Published: July 2010

Abstract: Autophosphorylation of Ca2+/calmodulin‐dependent protein kinase II (CaMKII) at Thr286 generates Ca2+‐independent activity. As an initial step toward understanding CaMKII inactivation, protein phosphatase classes (PP1, PP2A, PP2B, or PP2C) responsible for dephosphorylation of Thr286 in rat forebrain subcellular fractions were identified using phosphatase inhibitors/activators, by fractionation using ion exchange chromatography and by immunoblotting. PP2A‐like enzymes account for >70% of activity toward exogenous soluble Thr286‐autophosphorylated CaMKII in crude cytosol, membrane, and cytoskeletal extracts; PP1 and PP2C account for the remaining activity. CaMKII is present in particulate fractions, specifically associated with postsynaptic densities (PSDs); each protein phosphatase is also present in isolated PSDs, but only PP1 is enriched during PSD isolation. When isolated PSDs dephosphorylated exogenous soluble Thr286‐autophosphorylated CaMKII, PP2A again made the major contribution. However, CaMKII endogenous to PSDs (32P autophosphorylated in the presence of Ca2+/calmodulin) was predominantly dephosphorylated by PP1. In addition, dephosphorylation of soluble and PSD‐associated CaMKII in whole forebrain extracts was catalyzed predominantly by PP2A and PP1, respectively. Thus, soluble and PSD‐associated forms of CaMKII appear to be dephosphorylated by distinct enzymes, suggesting that Ca2+‐independent activity of CaMKII is differentially regulated by protein phosphatases in distinct subcellular compartments.
An Adenoviral Vector‐Based System to Study Neuronal Gene Expression: Analysis of the Rat Tyrosine Hydroxylase Promoter in Cultured Neurons
Authors: Robert Jean‐Jacques , Geoffroy Marie‐Claude , Finiels Françoise , Mallet Jacques
Published: July 2010

Abstract: We validated an adenoviral vector‐based system as a move toward the characterization of regulatory sequences that are involved in the control of cell‐type specificity and ligand regulation of neuronal gene expression in cultured neurons. We constructed recombinant adenoviruses, incorporating the luciferase gene under the control of different fragments of the rat tyrosine hydroxylase (TH) promoter. Similar results for luciferase expression were obtained in immortalized cells either by infection using adenoviral constructs or by transfection using conventional plasmid vectors. Taking advantage of adenoviral vectors, we extended our experiments to various primary cell cultures. The first 800 bp of the TH promoter were found to be sufficient to confer a cell‐type preferential activity in noradrenergic neurons of the rat superior cervical ganglia. Furthermore, using this neuronal culture model, we showed that the same promoter region carries leukemia‐inhibitory factor (LIF)‐responsive element(s). Our results demonstrate that the first 800 bp of the rat TH promoter contains a functionally important core region for constitutive and LIF‐regulated expression of TH in peripheral noradrenergic neurons. Moreover, the study validates the adenoviral vector‐based system as a new strategy for studying the regulation of neuronal gene expression.
Differential Expression of Multiple Somatostatin Receptors in the Rat Cerebellum During Development
Authors: Viollet C. , Bodenant C. , Prunotto C. , Roosterman D. , Schaefer J. , Meyerhof W. , Epelbaum J. , Vaudry H. , Leroux P.
Published: July 2010

Abstract: The present study describes the expression pattern of somatostatin receptor genes during the development of rat cerebellum. Characterization of somatostatin receptors was carried out by binding studies using receptor subtype‐selective ligands in the germinative epithelium and granule cell layer of the cerebellum from postnatal day 4 (P4) to P21 and in granule cell cultures. Quantitative reverse transcription‐PCR carried out for the five receptor subtype mRNAs in cerebellar extracts showed that sst1 mRNAs are predominant at the end of gestation. A transient high expression of the sst2 gene was observed from P7 to P14. In parallel, high levels of binding sites sensitive to sst2 ligands were detected in the granule cell germinative epithelium and in granule cell cultures. sst3 mRNAs rapidly increased from P14 and became the predominant form at P21, but respective binding sites were not detected. Whereas sst4 mRNA levels were generally low, those of sst5 were nearly undetectable. Reverse transcription‐PCR carried out in granule cell cultures revealed the relative abundance of sst mRNAs as follows: sst2 > sst1 > sst3 = sst4. sst5 mRNA was undetectable. The results show the expression of four somatostatin receptor genes, but only three receptors (sst1, sst4, and mainly sst2) were detected as binding sites during cerebellar development.
Depolarization and Activation of Dihydropyridine‐Sensitive Ca2+ Channels Stimulate Inositol Phosphate Accumulation in Photoreceptor‐Enriched Chick Retinal Cell Cultures
Authors: Gan Jiwei , Iuvone P. Michael
Published: July 2010

Abstract: Elevated concentrations of extracellular K+ increased inositol phosphate accumulation in primary cultures of chick retinal photoreceptors and multipolar neurons. K+‐evoked stimulation of inositol phosphate accumulation was greater in photoreceptor‐enriched cell cultures than in cultures where multipolar neurons were the predominant cell type. Destroying multipolar neurons, but not photoreceptors, with kainic acid and N‐methyl‐d‐aspartate did not reduce the K+‐evoked stimulation of inositol phosphate accumulation. Both of these observations indicate that the observed effects occur in photoreceptor cells. The K+‐evoked stimulation of inositol phosphate accumulation was blocked by omitting Ca2+ from the incubation medium or by adding the dihydropyridine‐sensitive Ca2+‐channel antagonists, nitrendipine and nifedipine. Bay K 8644, a dihydropyridine agonist, stimulated inositol phosphate accumulation and enhanced the effect of K+. ω‐Conotoxin GVIA, an inhibitor of N‐type Ca2+ channels, had no significant effect on K+‐stimulated inositol phosphate accumulation. Pretreatment with pertussis toxin neither blocked K+‐evoked inositol phosphate accumulation nor altered the inhibitory effect of nifedipine. K+‐evoked inositol phosphate accumulation appears to reflect activation of phosphatidylinositol‐specific phospholipase C, as it is inhibited by U‐73122. These results indicate that Ca2+ influx through voltage‐gated, dihydropyridine‐sensitive channels activates phospholipase C in photoreceptor inner segments and/or synaptic terminals.
Comparison of Type 2 Inositol 1,4,5‐Trisphosphate Receptor Distribution and Subcellular Ca2+ Release Sites that Support Ca2+ Waves in Cultured Astrocytes
Authors: Sheppard Carol A. , Simpson Peter B. , Sharp Alan H. , Nucifora Frederick C. , Ross Christopher A. , Lange G. David , Russell James T.
Published: July 2010

Abstract: We have examined the mechanisms that underlie Ca2+ wave propagation in cultured cortical astrocytes. Norepinephrine evoked Ca2+ waves in astrocytes that began at discrete initiation loci and propagated throughout the cell by regenerative amplification at a number of cellular sites, as shown by very high Ca2+ release rates at these regions. We have hypothesized previously that domains displaying elevated Ca2+ release kinetics in astrocytes may correspond to sites of high inositol 1,4,5‐trisphosphate receptor (InsP3R) density. To examine this possibility, we compared the distribution pattern of endoplasmic reticulum (ER) and InsP3Rs with Ca2+ release kinetics in subcellular regions during propagation of norepinephrine‐evoked waves. 3,3′‐Dihexyloxacarbocyanine iodide staining revealed that the ER in astrocytes exists as a meshwork of membranes extending throughout the cells, including fine processes. A specific antibody directed against type 2 InsP3Rs (InsP3R2) detected a 260‐kDa band in western blotting of astrocyte membranes. Immunocytochemistry using this antibody stained the entire ER system in a punctate, variegated manner. When Ca2+ responses and InsP3R2 immunofluorescence were compared in the same cell, domains of elevated Ca2+ response kinetics (high amplitude and rapid rate of rise) showed significant positive correlation with high local intensity of InsP3R2 staining. It appears, therefore, that specializations in the ER responsible for discrete local Ca2+ release sites that support regenerative wave propagation include increased levels of InsP3R2 expression.
Chronic Exposure to κ‐Opioids Enhances the Susceptibility of Immortalized Neurons (F‐11κ7) to Apoptosis‐Inducing Drugs by a Mechanism that May Involve Ceramide
Authors: Dawson G. , Dawson S. A. , Goswami R.
Published: July 2010

Abstract: Chronic exposure of embryonic brain to opioids leads to microcephaly and developmental abnormalities. An immortalized mouse neuroblastoma × dorsal root ganglion hybrid cell line stably transfected to overexpress κ‐opioid receptors (F‐11κ7) showed complete loss of κ‐receptor binding to [3H]U69,593 after exposure to the κ‐agonist U69,593 for 24 h. U69,593 had no measurable effect on cell viability as determined by either cell viability or DNA fragmentation assays. However, when cell death (apoptosis) was induced by either staurosporine or the phosphatidylinositol 3‐kinase inhibitors wortmannin and LY294002, cells pretreated with U69,593 for 24 h showed increased apoptosis compared with untreated cells. Thus, staurosporine (50 nM), wortmannin (4 µM), and LY294002 (30 µM) treatment for 24 h induced a 50% loss of cell viability and DNA fragmentation in 24 h. U69,593 pretreatment produced the same killing at lower concentrations, namely, 20 nM staurosporine, 2 µM wortmannin, and 14 µM LY294002, respectively. The effects of U69,593 were time‐, dose‐, and naloxone‐reversible, suggesting that they are receptor‐mediated. However, coaddition of U69,593 at the same time as staurosporine, wortmannin, or LY294002 did not enhance apoptosis. All three drugs that induced apoptosis were found to increase the level of ceramide, and pretreatment with U69,593 further increased the rate of formation of ceramide, a lipid that induces apoptosis in cells. We propose that chronic exposure to κ‐receptor agonists promotes increased vulnerability of neurons to apoptosis.
Cerebral Astrocytes Transport Ascorbic Acid and Dehydroascorbic Acid Through Distinct Mechanisms Regulated by Cyclic AMP
Authors: Siushansian Ramin , Tao Lei , Dixon S. Jeffrey , Wilson John X.
Published: July 2010

Abstract: Cerebral ischemia and trauma lead to rapid increases in cerebral concentrations of cyclic AMP and dehydroascorbic acid (DHAA; oxidized vitamin C), depletion of intracellular ascorbic acid (AA; reduced vitamin C), and formation of reactive astrocytes. We investigated astrocytic transport of AA and DHAA and the effects of cyclic AMP on these transport systems. Primary cultures of astrocytes accumulated millimolar concentrations of intracellular AA when incubated in medium containing either AA or DHAA. AA uptake was Na+‐dependent and inhibited by 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid (DIDS), whereas DHAA uptake was Na+‐independent and DIDS‐insensitive. DHAA uptake was inhibited by cytochalasin B, d‐glucose, and glucose analogues specific for facilitative hexose transporters. Once inside the cells, DHAA was reduced to AA. DHAA reduction greatly decreased astrocytic glutathione concentration. However, experiments with astrocytes that had been previously depleted of glutathione showed that DHAA reduction does not require physiological concentrations of glutathione. Astrocyte cultures were treated with a permeant analogue of cyclic AMP or forskolin, an activator of adenylyl cyclase, to induce cellular differentiation and thus provide in vitro models of reactive astrocytes. Cyclic AMP stimulated uptake of AA, DHAA, and 2‐deoxyglucose. The effects of cyclic AMP required at least 12 h and were inhibited by cycloheximide, consistent with a requirement for de novo protein synthesis. Uptake and reduction of DHAA by astrocytes may be a recycling pathway that contributes to brain AA homeostasis. These results also indicate a role for cyclic AMP in accelerating the clearance and detoxification of DHAA in the brain.
Stable Expression of Recombinant AMPA Receptor Subunits: Binding Affinities and Effects of Allosteric Modulators
Authors: Hennegriff Martin , Arai Amy , Kessler Markus , Vanderklish Peter , Mutneja Manpreet Singh , Rogers Gary , Neve Rachael L. , Lynch Gary
Published: July 2010

Abstract: Homomeric AMPA (α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid)‐type glutamate receptors (GluRs) were stably expressed in kidney cells from cDNAs encoding GluR1 flop, GluR2 flip, GluR2 flop, and GluR3 flop subunits. The recombinant receptors were of the expected size and showed functional properties in whole‐cell recording as previously reported. [3H]AMPA binding to all subunits was increased to a similar extent by the chaotropic ion thiocyanate (SCN−). Significant differences were found in the Scatchard plots, however, which were linear and of high affinity for GluR1 and ‐3 receptors (KD values of 33 and 52 nM, respectively) but showed curvature for GluR2 receptors, indicating the presence of two components with distinct affinities. As with brain AMPA receptors, solubilization of GluR2 receptors reduced the number of lower‐affinity sites and correspondingly increased the number of higher‐affinity sites. The sulfhydryl reagent p‐chloromercuriphenylsulfonic acid, which increases binding to brain receptors, produced only minor changes except in the case of GluR2 flip. These results indicate that GluR2, among the subunits examined here, most closely resembles the native AMPA receptors in brain membranes. [3H]AMPA binding was inhibited in a noncompetitive manner by two drugs that change the desensitization kinetics of the AMPA receptor. In agreement with physiological observations, the apparent affinity of cyclothiazide for GluR2 flip (EC50 = 7 µM) was higher than that for receptors made of flop subunits (49–130 µM). In contrast, BDP‐37, a member of the benzamide family of drugs, exhibited a lower potency for GluR2 flip (58 µM) than for any of the flop isoforms (18–40 µM). These results predict that the action of centrally active AMPA‐receptor modulators varies across brain regions depending on their flip/flop composition.
Cocaine Induces Apoptosis in Cortical Neurons of Fetal Mice
Authors: Nassogne Marie‐Cécile , Louahed Jamila , Evrard Philippe , Courtoy Pierre J.
Published: July 2010

Abstract: Exposure of fetal mouse brain cocultures to cocaine results selectively in the loss of neurites followed by neuronal death. By using enriched neuronal cultures, we here demonstrate that disappearance of neurons, when cultured with cocaine, is caused by apoptosis, based on (1) characteristic morphology of apoptotic nuclei at the level of neurons but not of glial cells by optic microscopy, and on total cell pellets by electron microscopy; (2) fragmentation of total DNA with a typical “ladder” pattern on agarose gels; (3) extensive in situ DNA fragmentation labeling (TUNEL method); and (4) prevention of cell loss by cycloheximide. The major metabolites of cocaine have no detectable effects on neurons, indicating that apoptosis is due to cocaine itself. Inappropriate neuronal apoptosis in cocaine‐exposed fetal brain could perturb the neurodevelopmental program and contribute to the quantitative neuronal defects that are too frequently reported in the offspring of cocaine‐abusing pregnant women.
Oxidatively Induced Structural Alteration of Glutamine Synthetase Assessed by Analysis of Spin Label Incorporation Kinetics: Relevance to Alzheimer's Disease
Authors: Butterfield D. Allan , Hensley Kenneth , Cole Pamela , Subramaniam Ramachandran , Aksenov Michael , Aksenova Marina , Bummer Paul M. , Haley Boyd E. , Carney John M.
Published: July 2010

Abstract: The activity of the astrocytic enzyme glutamine synthetase (GS) is decreased in the Alzheimer's disease brain, which may have relevance to mechanisms of chronic excitotoxicity. The molecular perturbation(s) that results in GS inactivation is not known, although oxidative lesioning of the enzyme is one likely cause. To assess structural perturbation induced in GS by metal‐catalyzed oxidation, a series of spin‐labeling studies were undertaken. Ovine GS was oxidized by exposure to iron/hydrogen peroxide and subsequently labeled with the thiol‐specific nitroxide probe MTS [(1‐oxyl‐2,2,5,5‐tetramethyl‐pyrroline‐3‐methyl)methanethiosulfonate]. The reaction of MTS with cysteine residues within GS was monitored in real time by electron paramagnetic resonance spectrometry. Structural perturbation of GS, manifested as decreased thiol accessibility, was inferred from an apparent decrease in the rate constant for the second‐order reaction of MTS with protein thiols. A subsequent spin‐labeling study was undertaken to compare the structural integrity of GS purified and isolated from Alzheimer's disease‐afflicted brain (AD‐GS) with that of GS isolated from nondemented, age‐matched control brain (C‐GS). The rate constant for reaction of MTS with AD‐GS was markedly decreased relative to that for the reaction of spin label with C‐GS. The kinetic data were partially corroborated by spectroscopic data obtained from circular dichroism analysis of control and peroxide‐treated ovine GS. In an adjunct experiment, the interaction of GS with a synthetic analogue of the Alzheimer's‐associated β‐amyloid peptide, known to induce free radical oxidative stress, indicated strong interaction of the enzyme with the peptide as reflected by a decrease in the rate constant for MTS binding to reactive protein thiols.
Inhibition of Tyrosine Aminotransferase by β‐N‐Oxalyl‐l‐α,β‐Diaminopropionic Acid, the Lathyrus sativus Neurotoxin
Authors: Vardhan K. Shasi , Rudra M. P. Pratap , Rao S. L. N.
Published: July 2010

Abstract: Species differences in susceptibility are a unique feature associated with the neurotoxicity of β‐N‐oxalyl‐l‐α,β‐diaminopropionic acid (l‐ODAP), the Lathyrus sativus neurotoxin, and the excitotoxic mechanism proposed for its mechanism of toxicity does not account for this feature. The present study examines whether neurotoxicity of l‐ODAP is the result of an interference in the metabolism of any amino acid and if it could form the basis to explain the species differences in susceptibility. Thus, Wistar rats and BALB/c (white) mice, which are normally resistant to l‐ODAP, became susceptible to it following pretreatment with tyrosine (or phenylalanine), exhibiting typical neurotoxic symptoms. C57BL/6J (black) mice were, however, normally susceptible to l‐ODAP without any pretreatment with tyrosine. Among the various enzymes associated with tyrosine metabolism examined, the activity of only tyrosine aminotransferase (TAT) was inhibited specifically by l‐ODAP. The inhibition was noncompetitive with respect to tyrosine (Ki = 2.0 ± 0.1 mM) and uncompetitive with respect to α‐ketoglutarate (Ki = 8.4 ± 1.5 mM). The inhibition of TAT was also reflected in a marked decrease in the rate of oxidation of tyrosine by liver slices, an increase in tyrosine levels of liver, and also a twofold increase in the dopa and dopamine contents of brain in l‐ODAP‐injected black mice. The dopa and dopamine contents in the brain of only l‐ODAP‐injected white mice did not show any change, whereas levels of these compounds were much higher in tyrosine‐pretreated animals. Also, the radioactivity associated with tyrosine, dopa, and dopamine arising from [14C]tyrosine was twofold higher in both liver and brain of l‐ODAP‐treated black mice. Thus, a transient increase in tyrosine levels following the inhibition of hepatic TAT by l‐ODAP and its increased availability for the enhanced synthesis of dopa and dopamine and other likely metabolites (toxic?) resulting therefrom could be the mechanism of neurotoxicity and may even underlie the species differences in susceptibility to this neurotoxin.
Chronic Intermittent Ethanol Treatment in Rats Increases GABAA Receptor α4‐Subunit Expression: Possible Relevance to Alcohol Dependence
Authors: Mahmoudi Mithra , Kang Maeng‐Hee , Tillakaratne Niranjala , Tobin Allan J. , Olsen Richard W.
Published: July 2010

Abstract: Chronic administration of ethanol to rats on an intermittent regimen, for 60 repeated intoxicating doses and repeated withdrawal episodes, results in a long‐lasting kindling phenomenon. This involves an increasing severity of withdrawal, including a reduced threshold to seizures produced by the GABAA antagonist, pentylenetetrazol. We have shown previously that muscimol‐evoked 36Cl− efflux and paired‐pulse inhibition (involving GABAA‐mediated recurrent inhibition) were decreased persistently in the CA1 region of hippocampal slices from chronic intermittent ethanol (CIE)‐treated rats. We now report elevated levels of mRNA in forebrain for the α4 subunit of the GABAA receptor (GABAR), considered to be a constituent of pharmacologically and physiologically novel subtypes of GABARs. Using in situ hybridization with digoxigenin‐labeled RNA probes, we show that at 2 days withdrawal, 60‐dose CIE leads to a significant 30% increase in α4 subunit mRNA levels in the dentate gyrus, 46% increase in the CA3, and 26% increase in the CA1 regions. In contrast, there was no significant change in the mRNAs for the α5 subunit or glutamic acid decarboxylase 67 in the same regions. This study suggests that GABAR subunit‐selective alterations occur after CIE treatment, possibly resulting in the alteration of the subunit composition of GABARs, with presumably altered physiological functions. This plasticity of GABARs may contribute to the increased withdrawal severity, reduced hippocampal inhibition, and increased seizure susceptibility of this animal model of human alcohol dependence.
Inhibition of Ischemia‐Induced Fodrin Breakdown by a Novel Phenylpyrimidine Derivative NS‐7: An Implication for Its Neuroprotective Action in Rats with Middle Cerebral Artery Occlusion
Authors: Takagaki Yumiko , Itoh Yoshinori , Aoki Yasuaki , Ukai Yojiro , Yoshikuni Yoshiaki , Kimura Kiyoshi
Published: July 2010

Abstract: The effect of a novel neuroprotective compound, NS‐7[4‐(4‐fluorophenyl)‐2‐methyl‐6‐(5‐piperidinopentyloxy)pyrimidine hydrochloride], on ischemia‐induced fodrin breakdown was examined both in vitro and in vivo. The fodrin breakdown was measured by western blot followed by a densitometric analysis. In slices of the rat cerebral cortex, a pronounced fodrin breakdown was observed under hypoxic and hypoglycemic conditions. The enhancement of fodrin breakdown was completely blocked by omission of extracellular Ca2+ and significantly inhibited by calpain inhibitors such as E‐64 and calpain inhibitor‐I, thereby suggesting that the fodrin breakdown induced by hypoxia/hypoglycemia is due to the activation of Ca2+‐stimulated neutral protease calpain. NS‐7 (1–30 µM) produced a concentration‐dependent inhibition of hypoxia/hypoglycemia‐induced fodrin breakdown. In rats with unilateral middle cerebral artery occlusion (MCAO), a pronounced fodrin breakdown was observed in the cerebral cortex and striatum, although the time course for the development of the fodrin breakdown was much slower in the cerebral cortex than in the striatum. NS‐7 (0.5 mg/kg i.v.), when injected immediately after MCAO, suppressed not only the fodrin breakdown but also the infarction in the cerebral cortex. From these results it is suggested that inhibition of calpain activation is implicated in the neuroprotective action of NS‐7.
Increased Secretion of the Amino‐Terminal Fragment of Amyloid Precursor Protein in Brains of Rats with a Constitutive Up‐Regulation of Protein Kinase C
Authors: Caputi A. , Barindelli S. , Pastorino L. , Cimino M. , Buxbaum J. D. , Cattabeni F. , Di Luca M.
Published: July 2010

Abstract: Protein kinase C (PKC) activation stimulates release of secreted amyloid precursor protein (APPs) in several cell lines. To ascertain the role of PKC in regulating APP metabolism in vivo, we used an animal model (methylazoxymethanol‐treated rats; MAM rats) in which PKC is permanently hyperactivated in selected brain areas, i.e., cortex and hippocampus. A significant decrease in membrane‐bound APP concentration was found in synaptosomes derived from cortex and hippocampus of MAM rats, where PKC is up‐regulated, with a concomitant increase in APPs production in soluble fractions of the same brain areas. In contrast, in a brain area not affected by MAM treatment (i.e., cerebellum), APP secretion is similar in control and MAM rats, indicating that altered metabolism of APP is restricted to only those areas in which the PKC system is up‐regulated. In addition, phorbol esters or H‐7 modulate APPs release in hippocampal slices from both control and MAM rats, further supporting an in vivo role for this enzyme in regulating metabolism of mature APP.
Spatial Learning Activates Neural Cell Adhesion Molecule Polysialylation in a Corticohippocampal Pathway Within the Medial Temporal Lobe
Authors: O'Connell Alan W. , Fox Gerard B. , Barry Thomas , Murphy Keith J. , Fichera Giacomo , Foley Andrew G. , Kelly John , Regan Ciaran M.
Published: July 2010

Abstract: Transient and time‐dependent modulations of neural cell adhesion molecule (NCAM) polysialylation in the dentate gyrus of the rodent hippocampus are a feature of spatial and nonspatial forms of learning. In the hippocampal formation, polysialic acid immunoreactivity was localized to granule‐like cells and their mossy fibre axons. We now demonstrate the latter to extend to the CA3 region where apparent recurrent and Schaffer collaterals were labelled. The axons of the CA1 pyramidal cell layer were immunopositive, as was the subiculum that they innervate. Layers I and III of the entorhinal cortex stained intensely for polysialic acid; however, these were not visible in the more lateral aspect of this region and were replaced by a single band of immunopositive neurons that extended to include the perirhinal and piriform cortices. After Morris water maze training, the number of polysialylated neurons within the entorhinal cortex exhibited a two‐ to threefold increase at the 10–12‐h posttraining time with respect to that observed immediately after training. This increase was task specific, as no change was observed in freely swimming animals or those required to locate a visible platform. These results suggest the presence of a corticohippocampal pathway involved in the eventual consolidation of memory.
Effects of Depolarization and NMDA Antagonists on the Survival of Cerebellar Granule Cells: A Pivotal Role for Protein Kinase C Isoforms
Authors: Lin Wan Wan , Wang Ching Wen , Chuang De‐Maw
Published: July 2010

Abstract: Primary cultures of cerebellar granule cells (CGCs) grown in high‐K+ (25 mM; K25) medium progressively differentiate in vitro. Differentiation is noticeable after 3–4 days in vitro (DIV) and reach a mature stage after 8 DIV. Longer cultivation of CGCs (>13 DIV) triggers the processes of spontaneous cell death. However, if cultured in normal physiological K+ concentration (5 mM; K5), a significant proportion of the cells dies by the end of the first week in culture. To address the role of protein kinase C (PKC) in the development of CGCs, we measured the kinase activity as well as the protein level of the kinase isoforms. As the K25 CGC culture proceeded, the PKC activity time‐dependently increased by 3.2‐fold, reaching a steady state at 8 DIV. Western blot analysis using PKC isoform‐specific antibodies revealed an increase in levels of PKC α, γ, μ, λ, and ι from 2 to 8 DIV. A slight increase or decrease at 4 DIV was observed for PKC ε and βII, respectively, whereas no significant change was observed for βI. The isoforms of δ, θ, η, and ζ were not detected. Comparing the 14 DIV cultures with the 10 DIV cultures, the immunoreactivities of PKC ι and ε were decreased, those of PKC α, βI, βII, γ, and λ were unchanged, whereas that of PKC μ was still increased. In K5 cultures, the immunoreactivity of each PKC isoform at 2–4 DIV was similar to that observed in K25 cells, although no remarkable differentiation features were observed. Coordinated with the appearance of cell death at 8 DIV in low‐K+ cultures, levels of PKC α, μ, λ, and ι, but not the others, were markedly decreased. The NMDA receptor antagonists MK‐801 and 2‐amino‐5‐phosphopentanoic acid markedly prevented the age‐induced apoptosis of CGCs, and the cells survived >18 DIV under these conditions. The cytoprotective effect of MK‐801 was concomitant with the increases in levels of PKC γ, λ, ι, and μ at 10 and 14 DIV. In addition, the PKC ε level was increased at 14 DIV but decreased at early stages, whereas PKC α, βI, and βII levels were unchanged, as compared with K25 culture alone. Taken together, induction and up‐regulation of PKC isoforms may play an important role in the maintenance of CGC survival by depolarization and MK‐801.
Effects of Wild‐Type and Mutated Copper/Zinc Superoxide Dismutase on Neuronal Survival and l‐DOPA‐Induced Toxicity in Postnatal Midbrain Culture
Authors: Mena Maria A. , Khan Uzma , Togasaki Daniel M. , Sulzer David , Epstein Charles J. , Przedborski Serge
Published: July 2010

Abstract: Mutations in the free radical‐scavenging enzyme copper/zinc superoxide dismutase (Cu/Zn‐SOD) are associated with neuronal death in humans and mice. Here, we examine the effects of human wild‐type (WT SOD) and mutant (Gly93→ Ala; G93A) Cu/Zn‐SOD enzyme on the fate of postnatal midbrain neurons. One‐week‐old cultures from transgenic mice expressing WT SOD enzyme had significantly more midbrain neurons and fewer necrotic and apoptotic neurons than non‐transgenic cultures. In contrast, 1‐week‐old cultures from transgenic G93A mice expressing mutant SOD enzyme had significantly fewer midbrain neurons and more necrotic and apoptotic neurons than nontransgenic cultures. To subject postnatal midbrain neurons to oxidative stress, cultures were incubated with l‐DOPA. l‐DOPA at 200 µM caused ∼50% loss of tyrosine hydroxylase (TH)‐positive neurons in nontransgenic cultures and even greater loss in transgenic G93A cultures; no alterations were noted in GABA neuron numbers. In contrast, 200 µMl‐DOPA did not cause any significant reductions in TH‐positive or GABA neuron numbers in transgenic WT SOD cultures. l‐DOPA at 50 µM had opposite effects, in that it significantly increased TH‐positive, but not GABA neuron numbers in transgenic WT SOD and G93A and in nontransgenic cultures. These results indicate that increased amounts of WT SOD enzyme promote cell survival and protect against l‐DOPA‐induced dopaminergic neurotoxicity, whereas increased amounts of mutated Cu/Zn‐SOD enzyme have inverse effects. As the spontaneous loss and l‐DOPA‐induced loss of postnatal dopaminergic midbrain neurons appear to be mediated by free radicals, our study supports the view that mutated Cu/Zn‐SOD enzyme kills cells by oxidative stress.
Distribution of Brain‐Derived Neurotrophic Factor in Rats and Its Changes with Development in the Brain
Authors: Katoh‐Semba Ritsuko , Takeuchi Ikuo K. , Semba Reiji , Kato Kanefusa
Published: July 2010

Abstract: A newly established, sensitive, two‐site enzyme‐immunoassay system for brain‐derived neurotrophic factor (BDNF) is described. Using this system, we investigated the tissue distribution of BDNF and developmental changes in tissue levels of BDNF in rats. The minimal limit of detection of the assay was 3 pg/0.2 ml of assay mixture. BDNF was successfully solubilized from tissues in the presence of guanidine hydrochloride but not in any of the other buffers examined. In the rat brain at 1 month of age, the highest level of BDNF was detected in the hippocampus (5.41 ng/g of wet weight), followed by the hypothalamus (4.23 ng/g) and the septum (1.68 ng/g). In other regions, levels of BDNF ranged between 0.9 and 1.7 ng/g. The level of BDNF in the posterior lobes of the cerebellum from rats at 30 days of age was slightly higher than that in the anterior lobes. The concentration of BDNF increased in all regions of the brain with postnatal development. In peripheral tissues, BDNF was found at very low concentrations (0.65 ng/g in the spleen, 0.21 ng/g in the thymus, and 0.06 ng/g in the liver). The subfractionation of the hippocampal homogenate indicated that ∼50% of BDNF was contained in the crude nuclear fraction. Immunoblots of BDNF‐immunoreactive proteins extracted from the hippocampus, hypothalamus, and cerebellum contained doublet bands of protein of ∼14 kDa, a value close to the molecular mass of recombinant human BDNF. Immunocytochemical investigations showed that, in the hippocampus, BDNF was localized in the nucleus of the granule cells in the dentate gyrus and of the cells in the pyramidal cell layer. The frequency of cells that were stained in the dentate gyrus was greater than that of cells in the pyramidal cell layer.
Dramatic Increase of the RNA Editing for Glutamate Receptor Subunits During Terminal Differentiation of Clonal Human Neurons
Authors: Lai Fang , Chen Chun‐Xia , Lee Virginia M.‐Y. , Nishikura Kazuko
Published: July 2010

Abstract: RNA editing plays an important role in determining physiological characteristics of certain glutamate‐gated receptor (GluR) channels such as Ca2+ permeability and desensitization kinetics. In one case, the editing changes a gene‐encoded glutamine (Q) to an arginine (R) codon located in the channel‐forming domain of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptor subunit GluR‐B and also the kainate receptor subunits GluR5 and GluR6. Another case of RNA editing alters an arginine (R) to a glycine (G) codon at a position termed the “R/G” site of AMPA subunits GluR‐B, C, and D. Double‐stranded RNA‐specific adenosine deaminases (DRADA) have been implicated as agents involved in the editing. By using a human teratocarcinoma cell line, NT2, we investigated the change of the RNA editing of GluR subunits in conjunction with the expression of two DRADA members, DRADA1 and DRADA2 genes, during neuronal differentiation. Whereas Q/R and R/G site RNA editing both become progressively activated in differentiating NT2 cells, the expression of the two DRADA genes can already be detected even in the undifferentiated NT2 cells. Development of the editing machinery appears to require, in addition to DRADA enzymes, a currently unidentified mechanism(s) that may become activated during neuronal differentiation.
Nerve Growth Factor Protects PC12 Cells Against Peroxynitrite‐Induced Apoptosis via a Mechanism Dependent on Phosphatidylinositol 3‐Kinase
Authors: Spear Nathan , Estévez Alvaro G. , Barbeito Luis , Beckman Joseph S. , Johnson Gail V. W.
Published: July 2010

Abstract: Nerve growth factor (NGF) prevents apoptosis induced by the oxidant peroxynitrite in undifferentiated PC12 rat pheochromocytoma cells. Previous studies have shown that activation of phosphatidylinositol 3‐kinase (PI 3‐kinase) by NGF via the TrkA receptor tyrosine kinase protects PC12 cells from serum deprivation‐induced apoptosis. We found that two PI 3‐kinase inhibitors, wortmannin and LY294002, eliminated the protection NGF provided against peroxynitrite‐induced apoptosis at concentrations consistent with their effectiveness as PI 3‐kinase inhibitors. When the activity of PI 3‐kinase was assayed in phosphotyrosine immunoprecipitates after treatment of PC12 cells with peroxynitrite, PI 3‐kinase activity was reduced by 50% of that detected in control cells, whereas PI 3‐kinase activity in NGF‐treated cells was unaffected by peroxynitrite. If an antibody against PI 3‐kinase was used to immunoprecipitate the enzyme, treatment with peroxynitrite had no effect on activity. Therefore, peroxynitrite appeared to disrupt interactions between PI 3‐kinase and phosphotyrosine proteins, rather than directly inhibiting the enzyme. NGF also activates p21Ras‐dependent pathways, but this did not appear to be required for NGF to exert its protective effect against peroxynitrite. PC12 cells expressing a dominant inhibitory mutant of p21Ras were equally susceptible to peroxynitrite‐induced apoptosis, which was prevented by NGF. Wortmannin was also able to block the protective effect of NGF in the p21Ras mutant cell line. Although many signaling pathways are activated by NGF, these results suggest that a PI 3‐kinase‐dependent pathway is important for inhibiting peroxynitrite‐induced apoptosis.
Lysophosphatidic Acid Induces a Sustained Elevation of Neuronal Intracellular Calcium
Authors: Holtsberg Frederick W. , Steiner Marion R. , Furukawa Katsutoshi , Keller Jeffrey N. , Mattson Mark P. , Steiner Sheldon M.
Published: July 2010

Abstract: Lysophosphatidic acid (LPA) is a lipid biomediator enriched in the brain. A novel LPA‐induced response in rat hippocampal neurons is described herein, namely, a rapid and sustained elevation in the concentration of free intracellular calcium ([Ca2+]i). This increase is specific, in that the related lipids phosphatidic acid and lysophosphatidylcholine did not induce an alteration in [Ca2+]i. Moreover, consistent with a receptor‐mediated process, there was no further increase in [Ca2+]i after a second addition of LPA. The LPA‐induced increase in [Ca2+]i required extracellular calcium. However, studies with Cd2+, Ni2+, and nifedipine and nystatin‐perforated patch clamp analyses did not indicate involvement of voltage‐gated calcium channels in the LPA‐induced response. In contrast, glutamate appears to have a significant role in the LPA‐induced increase in [Ca2+]i, because this increase was inhibited by NMDA receptor antagonists and α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)/kainate receptor antagonists. Thus, LPA treatment may result in an increased extracellular glutamate concentration that could stimulate AMPA/kainate receptors and thereby alleviate the Mg2+ block of the NMDA receptors and lead to glutamate stimulation of an influx of calcium via NMDA receptors.