bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of the GABAA Receptor‐Associated Protein (GABARAP) to Microtubules and Microfilaments Suggests Involvement of the Cytoskeleton in GABARAPGABAA Receptor Interaction
Authors: Wang Hongbing , Olsen Richard W
Published: March 2003

GABAA receptor‐associated protein (GABARAP) was isolated on the

basis of its interaction with the γ2 subunit of GABAA

receptors. It has sequence similarity to light chain 3 (LC3) of

microtubule‐associated proteins 1A and 1B. This suggests that GABARAP may link

GABAA receptors to the cytoskeleton. GABARAP associates with

tubulin in vitro. However, little is known about the mechanism for the

interaction, and it is not clear whether the interaction occurs in vivo. Here,

we report that GABARAP interacts directly with both tubulin and microtubules

in a salt‐sensitive manner, indicating the association is mediated by ionic

interactions. GABARAP coimmunoprecipitates with tubulin and associates with

both microtubules and microfilaments in intact cells. The cellular

distribution is altered by treatment with taxol, nocodazole, and cytochalasin

D. The tubulin binding domain was located at the N terminus of GABARAP by

using synthetic peptides and deletion constructs and is marked by a specific

arrangement of basic amino acids. The interaction between GABARAP and actin

might be mediated by other proteins. These results demonstrate the GABARAP

interacts with the cytoskeleton both in vitro and in cells and suggest a role

of GABARAP in the interaction between GABAA receptors and the cytoskeleton. Such interactions are presumably needed for receptor trafficking, anchoring, and/or synaptic clustering. The structural arrangement of the basic amino acids present in the tubulin binding domain of GABARAP may aid in recognition of the potential of tubulin binding activity in other known proteins.
Calmodulin Regulation of Basal and Agonist‐Stimulated G Protein Coupling by the μ‐Opioid Receptor (OP3) in Morphine‐Pretreated Cell
Authors: Wang Danxin , Surratt Christopher K , Sadée Wolfgang
Published: March 2003

Calmodulin (CaM) has been shown to suppress basal G protein coupling and attenuate agonist‐stimulated G protein coupling of the μ‐opioid receptor (OP3) through direct interaction with the third intracellular (i3)

loop of the receptor. Here we have investigated the role of CaM in regulating

changes in OP3‐G protein coupling during morphine treatment, shown

to result in CaM release from plasma membranes. Basal and agonist‐stimulated G

protein coupling by OP3 was measured before and after morphine

pretreatment by incorporation of guanosine

5′‐O‐(3‐[35S]thiotriphosphate) into membranes,

obtained from HEK 293 cells transfected with human OP3 cDNA. The

opioid antagonist β‐chlornaltrexamine fully suppressed basal G protein

coupling of OP3, providing a direct measure of basal signaling.

Pretreatment of the cells with morphine enhanced basal G protein coupling

(sensitization). In contrast, agonist‐stimulated coupling was diminished

(desensitization), resulting in a substantially flattened morphine

dose‐response curve. To test whether CaM is involved in these changes, we

constructed OP3‐i3 loop mutants with reduced affinity for CaM

(K273A, R275A, and K273A/R275A). Basal signaling of these mutant

OP3 receptors was higher than that of the wild‐type receptor and,

moreover, unaffected by morphine pretreatment, whereas desensitization to

agonist stimulation was only slightly attenuated. Therefore,

CaM‐OP3 interactions appear to play only a minor role in the

desensitization of OP3. In contrast, release of CaM from the plasma

membrane appears to enhance the inherent basal G protein coupling of

OP3, thereby resolving the paradox that OP3 displays both desensitization and sensitization during morphine treatment.
Extracellular ATP Triggers Tumor Necrosis Factor‐α Release from Rat Microglia
Authors: Hide Izumi , Tanaka Masaya , Inoue Atsuko , Nakajima Kazuyuki , Kohsaka Shinichi , Inoue Kazuhide , Nakata Yoshihiro
Published: March 2003

Abstract: Brain microglia are a major source of inflammatory

cytokines, such as tumor necrosis factor‐α (TNF‐α), which have

been implicated in the progression of neurodegenerative diseases. Recently,

microglia were revealed to be highly responsive to ATP, which is released from

nerve terminals, activated immune cells, or damaged cells. It is not clear,

however, whether released ATP can regulate TNF‐α secretion from

microglia. Here we demonstrate that ATP potently stimulates TNF‐α

release, resulting from TNF‐α mRNA expression in rat cultured brain

microglia. The TNF‐α release was maximally elicited by 1 mM ATP

and also induced by a P2X7 receptor‐selective agonist, 2′‐

and 3′‐O‐(4‐benzoylbenzoyl)adenosine 5′‐triphosphate,

suggesting the involvement of P2X7 receptor. ATP‐induced

TNF‐α release was Ca2+‐dependent, and a sustained

Ca2+ influx correlated with the TNF‐α release in

ATP‐stimulated microglia. ATP‐induced TNF‐α release was inhibited by PD

098059, an inhibitor of extracellular signal‐regulated protein kinase (ERK)

kinase 1 (MEK1), which activates ERK, and also by SB 203580, an inhibitor of

p38 mitogen‐activated protein kinase. ATP rapidly activated both ERK and p38

even in the absence of extracellular Ca2+. These results indicate

that extracellular ATP triggers TNF‐α release in rat microglia via a P2

receptor, likely to be the P2X7 subtype, by a mechanism that is

dependent on both the sustained Ca2+ influx and ERK/p38 cascade,

regulated independently of Ca2+ influx.
Functional Comparison of Egr3 Transcription Factor Isoforms
Authors: O'Donovan Kevin J. , Levkovitz Yechiel , Ahn David , Baraban Jay M.
Published: March 2003

Abstract: Recent studies indicate that the Egr family of
transcription regulatory factors plays a key role in nervous system
development and plasticity. In prior studies, we demonstrated that multiple
isoforms of the Egr3 transcription regulatory factor are expressed in brain
and appear to be generated by use of alternative translation start sites. To
compare the functional activity of these isoforms, we have examined their
ability to stimulate transcription of a luciferase reporter construct driven
by the Egr response element. Analysis of a series of N‐terminal truncation
constructs indicates that Egr3 contains two distinct activation domains: one
located in the segment upstream of Met106, the start site of the
major truncated isoform Egr3β, and the other located C‐terminal to all of
the alternative translation start sites used to generate Egr3 isoforms
detected in brain. We confirmed this inference by demonstrating that each of
these segments is able to drive transcription when fused to the GAL4 DNA
binding domain. Taken together, these studies indicate that the internal
translation start sites present in Egr3 are used to generate Egr3 isoforms
lacking the activation domain located N‐terminal to Met106.
Circadian Binding Activity of AP‐1, a Regulator of the Arylalkylamine N‐Acetyltransferase Gene in the Rat Pineal Gland, Depends on Circadian Fra‐2, c‐Jun, and Jun‐D Expression and Is Regulated by the Clock's Zeitgebers
Authors: Guillaumond F. , Sage D. , Deprez P. , Bosler O. , Becquet D. , François‐Bellan A. M.
Published: March 2003

Abstract: The daily rhythm in circulating melatonin is driven by a
circadian rhythm in the expression of the arylalkylamine
N‐acetyltransferase gene in the rat pineal gland. Turning off
expression of this gene at the end of night is believed to involve inhibitory
transcription factors, among which Fos‐related antigen 2 (Fra‐2) appears as a
good candidate. Circadian rhythms in the expression of three proteins of
activating protein‐1 (AP‐1) complexes, namely, Fra‐2, c‐Jun, and Jun‐D, are
shown here to account for circadian variations in AP‐1 binding activity.
Quantitative variations in the Fra‐2 component over the circadian cycle were
associated with qualitative variations in protein isoforms. Destruction of the
suprachiasmatic nucleus resulted in decreased nocturnal AP‐1 activity, showing
that AP‐1 circadian rhythm is driven by this nucleus. Exposure to light during
subjective night and administration of a serotonin
5‐HT1A/5‐HT7 receptor agonist during subjective day, respectively, induced a 50% decrease and a 50% increase in both AP‐1 and Fra‐2 expression. These effects were impaired by suprachiasmatic nucleus lesions. These data show that pineal AP‐1 binding activity, which results from Fra‐2 expression, can be modulated by light and serotonin through the suprachiasmatic nucleus according to a “phase dependence” that is characteristic of the rhythm of clock sensitivity to both zeitgebers.
Gliotoxic Action of Glutamate on Cultured Astrocytes
Authors: Chen Chun‐Jung , Liao Su‐Lan , Kuo Jon‐Son
Published: March 2003

Abstract: Because of the well‐documented importance of glutamate clearance by astrocytes in protecting neurons against excitotoxicity, it was interesting to examine whether L‐glutamate exerts a toxic action on cultured astrocytes. Cell damage was evaluated by measuring activity of lactate dehydrogenase (LDH) released into the culture medium. Exposure of astrocyte cultures of the neonatal rat cerebral cortex to L‐glutamate resulted in a concentration‐ and time‐dependent increase in the release of LDH. L‐Glutamate‐induced gliotoxicity appeared to be mediated predominantly by the increase of oxidative stress because the reduced glutathione content and its effects were almost completely blocked by vitamin E and pyrrolidinedithiocarbamate. To support this notion further, the supplementation or depletion of intracellular reduced glutathione content attenuated or worsened L‐glutamate toxicity, respectively. Activation of the glutamate transporter mimicked the action of L‐glutamate on astrocytes. In addition, degrees of cell damage were not directly correlated to the levels of glutamate uptake. Moreover, the mechanism of this toxicity required energy and macromolecular synthesis. Taken together, brief exposure to L‐glutamate resulted in glutamate uptake and cell swelling, whereas sustained exposure injured astrocytes via oxidative stress instead of the excitatory mechanism.
Unique Functional Properties of a Sensory Neuronal P2X ATP‐Gated Channel from Zebrafish
Authors: Boué‐Grabot Éric , Akimenko Marie‐Andrée , Séguéla Philippe
Published: March 2003

Abstract: We report here the structural and functional
characterization of an ionotropic P2X ATP receptor from the lower vertebrate
zebrafish (Danio rerio). The full‐length cDNA encodes a 410‐amino
acid‐long channel subunit zP2X3, which shares only 54% identity
with closest mammalian P2X subunits. When expressed in Xenopus
oocytes in homomeric form, ATP‐gated zP2X3 channels evoked a unique
nonselective cationic current with faster rise time, faster kinetics of
desensitization, and slower recovery than any other known P2X channel.
Interestingly, the order of agonist potency for this P2X receptor was found
similar to that of distantly related P2X7 receptors, with
benzoylbenzoyl ATP (EC50 = 5 μM) ≫ ATP
(EC50 = 350 μM) = ADP > α,β‐methylene ATP
(EC50 = 480 μM). zP2X3 receptors are highly
sensitive to blockade by the antagonist trinitrophenyl ATP (IC50
< 5 nM) but are weakly sensitive to the noncompetitive antagonist
pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid.
zP2X3 subunit mRNA is exclusively expressed at high levels in trigeminal neurons and Rohon‐Beard cells during embryonic development, suggesting that neuronal P2X receptors mediating fast ATP responses were selected early in the vertebrate phylogeny to play an important role in sensory pathways.
Inhibition of Tyrosinase Reduces Cell Viability in Catecholaminergic Neuronal Cells
Authors: Higashi Youichirou , Asanuma Masato , Miyazaki Ikuko , Ogawa Norio
Published: March 2003

Abstract: The biosynthesis of dopamine (DA) in catecholaminergic neurons is regulated by tyrosine hydroxylase, which converts tyrosine into 3,4‐dihydroxyphenylalanine (L‐DOPA). In melanocytes, tyrosinase catalyzes both the hydroxylation of tyrosine and the consequent oxidation of L‐DOPA to form melanin. Although it has been demonstrated that tyrosinase is also expressed in the brain, the physiological role of tyrosinase in the brain is still obscure. In this study, to investigate the role of tyrosinase in catecholaminergic neuronal cells, we examined the effects of tyrosinase inhibition on the viability of CATH.a and SH‐SY5Y cells using tyrosinase inhibitors—specifically, phenylthiourea (PTU) and 5‐hydroxyindole (5‐HI)—and the transfection of antisense tyrosinase cDNA. Both inhibitors significantly reduced the cell viability of CATH.a cells in a dose‐dependent manner. PTU also specifically enhanced DA‐induced cell death, but 5‐HI did not. This discrepancy in cell death is probably due to the inhibitors' different mechanism of action: 5‐HI inhibits the hydroxylation of tyrosine as a competitor for the substrate to induce cell death that may be due to depletion of DA, whereas PTU mainly inhibits the enzymatic oxidation of L‐DOPA and DA rather than tyrosine hydroxylation to increase consequently autooxidation of DA. Indeed, the intracellular DA content in CATH.a cells was enhanced by PTU exposure. In contrast, PTU showed no enhancing effects on DA‐induced cell death of SH‐SY5Y cells, which express little tyrosinase. Furthermore, transfection with antisense tyrosinase cDNA into CATH.a cells dramatically reduced cell viability and significantly enhanced DA‐induced cell death. These results suggest that tyrosinase controls the intracellular DA content by biosynthesis or enzymatic oxidation of DA, and the dysfunction of this activity induces cell death by elevation of intracellular DA level and consequent gradual autooxidation of DA to generate reactive oxygen species.
Electrically Evoked Release of [3H]Noradrenaline from Mouse Cultured Sympathetic Neurons
Authors: Göbel Ingrid , Trendelenburg Anne Ulrike , Cox Sandra L. , Meyer Angelika , Starke Klaus
Published: March 2003

Abstract: Cultured neurons from the thoracolumbar sympathetic chain
of newborn mice are known to possess release‐inhibiting
α2‐autoreceptors. The present study was carried out in a
search for release‐modulating heteroreceptors on these neurons. Primary
cultures were preincubated with [3H]noradrenaline and then
superfused and stimulated by single pulses, trains of 8 pulses at 100 Hz, or
trains of 36 pulses at 3 Hz. The cholinergic agonist carbachol reduced the
evoked overflow of tritium. Experiments with antagonists indicated that the
inhibition was mediated by M2 muscarinic receptors. The cannabinoid
agonist WIN 55,212‐2 reduced the evoked overflow of tritium through
CB1 receptors. Prostaglandin E2, sulprostone, and
somatostatin also caused presynaptic inhibition. The inhibitory effects of
carbachol, WIN 55,212‐2, prostaglandin E2, and somatostatin were
abolished (at the highest concentration of WIN 55,212‐2 almost abolished) by
pretreatment of the cultures with pertussis toxin (250 ng/ml). Several drugs,
including the β2‐adrenoceptor agonist salbutamol, opioid receptor agonists, neuropeptide Y, angiotensin II, and bradykinin, failed to change the evoked overflow of tritium. These results demonstrate a distinct pattern of presynaptic inhibitory heteroreceptors, all coupled to pertussis toxin‐sensitive G proteins. The lack of operation of several presynaptic receptors known to exist in adult mice in situ may be due to the age of the (newborn) donor animals or to the culture conditions.
5‐HT1B Receptor‐Mediated Regulation of Serotonin Clearance in Rat Hippocampus In Vivo
Authors: Daws Lynette C. , Gould Georgianna G. , Teicher Susan D. , Gerhardt Greg A. , Frazer Alan
Published: March 2003

Abstract: The 5‐hydroxytryptamine (5‐HT; serotonin) transporter
(5‐HTT) is important in terminating serotonergic neurotransmission and is a
primary target for many psychotherapeutic drugs. Study of the regulation of
5‐HTT activity is therefore important in understanding the control of
serotonergic neurotransmission. Using high‐speed chronoamperometry, we have
demonstrated that local application of 5‐HT1B antagonists into the
CA3 region of the hippocampus prolongs the clearance of 5‐HT from
extracellular fluid (ECF). In the present study, we demonstrate that the
5‐HT1B antagonist cyanopindolol does not produce this effect by
increasing release of endogenous 5‐HT or by directly binding to the 5‐HTT.
Dose‐response studies showed that the potency of cyanopindolol to inhibit
clearance of 5‐HT was equivalent to that of the selective 5‐HT reuptake
inhibitor fluvoxamine. Local application of the 5‐HT1A antagonist
WAY 100635 did not alter 5‐HT clearance, suggesting that the effect of
cyanopindolol to prolong clearance is not via a mechanism involving
5‐HT1A receptors. Finally, the effect of low doses of cyanopindolol
and fluvoxamine to inhibit clearance of 5‐HT from ECF was additive. These data
are consistent with the hypothesis that activation of terminal
5‐HT1B autoreceptors increases 5‐HTT activity.
The A‐Type Potassium Channel Kv4.2 Is a Substrate for the Mitogen‐Activated Protein Kinase ERK
Authors: Adams J. Paige , Anderson Anne E. , Varga Andrew W. , Dineley Kelly T. , Cook Richard G. , Pfaffinger Paul J. , Sweatt J. David
Published: March 2003

Abstract: The mitogen‐activated protein kinase ERK has recentlybecome a focus of studies of synaptic plasticity and learning and memory. Dueto the prominent role of potassium channels in regulating the electricalproperties of membranes, modulation of these channels by ERK could play animportant role in mediating learning‐related synaptic plasticity in the CNS.Kv4.2 is a Shal‐type potassium channel that passes an A‐type current and islocalized to dendrites and cell bodies in the hippocampus. The sequence ofKv4.2 contains several consensus sites for ERK phosphorylation. In the presentstudies, we tested the hypothesis that Kv4.2 is an ERK substrate. Wedetermined that the Kv4.2 C‐terminal cytoplasmic domain is an effective ERK2substrate, and that it is phosphorylated at three sites: Thr602,Thr607, and Ser616. We used this information to developantibodies that recognize Kv4.2 phosphorylated by ERK2. One of ourphospho‐site‐selective antibodies was generated using a triply phosphorylatedpeptide as the antigen. We determined that this antibody recognizesERK‐phosphorylated Kv4.2 in COS‐7 cells transfected with Kv4.2 and nativeERK‐phosphorylated Kv4.2 in the rat hippocampus. These observations indicatethat Kv4.2 is a substrate for ERK in vitro and in vivo, and suggest that ERKmay regulate potassium‐channel function by direct phosphorylation of thepore‐forming α subunit.
Regulation of Phospholipase D2 by H2O2 in PC12 Cells
Authors: Oh Sae‐Ock , Hong Jang‐Hee , Kim Young‐Rae , Yoo Hyang‐Sook , Lee Sang‐Ho , Lim Kyu , Hwang Byung‐Doo , Exton John H. , Park Seung‐Kiel
Published: March 2003

Abstract: Phospholipase D2 (PLD2) is expressed in brain andinhibited by synuclein, which is involved in Parkinson's and Alzheimer'sdiseases. However, the activation mechanism of PLD2 in neuronal cells has notbeen defined clearly. Hydrogen peroxide (H2O2) playsroles in the neurodegenerative diseases and also acts as a second messenger ofvarious molecules such as nerve growth factor. To study regulation mechanismsof PLD2 by H2O2 in neuronal cells, we have made stablePC12 cell lines expressing PLD2 (PLD2‐PC12 cells). H2O2treatment stimulated PLD activity in PLD2‐PC12 cells in a dose‐ andtime‐dependent manner. This activation was inhibited by the treatment withprotein kinase C (PKC) inhibitors or by depletion of PKCα, ‐δ, and‐ε. Phorbol ester markedly activated PLD2. Co‐treatment with phorbolester and H2O2 did not show an additive effect.Chelation of extracellular calcium substantially blocked theH2O2‐induced activation of PLD2. A calcium ionophoreinduced PLD2 activation in a PKC‐dependent manner. Protein‐tyrosine kinaseinhibitors inhibited H2O2‐induced PLD activationslightly. These data indicate that H2O2 can activatePLD2 in PC12 cells and that this activation is largely dependent on PKC andCa2+ ions and minimally dependent on tyrosine phosphorylation.
Characterisation of N‐Methyl‐D‐Aspartate Receptor‐Specific [3H]Ifenprodil Binding to Recombinant Human NR1a/NR2B Receptors Compared with Native Receptors in Rodent Brain Membranes
Authors: Grimwood Sarah , Richards Pawel , Murray Fraser , Harrison Neil , Wingrove Peter B. , Hutson Peter H.
Published: March 2003

Abstract: We have performed [3H]ifenprodil bindingexperiments under NMDA receptor‐specific assay conditions to provide the firstdetailed characterisation of the pharmacology of the ifenprodil site on NMDANR1/NR2B receptors, using recombinant human NR1a/NR2B receptors stablyexpressed in L(tk‐) cells, in comparison with rat cortex/hippocampusmembranes. [3H]Ifenprodil bound to a single, saturable site on bothhuman recombinant NR1a/NR2B receptors and native rat receptors withBmax values of 1.83 and 2.45 pmol/mg of protein,respectively, and KD values of 33.5 and 24.8 nM,respectively. The affinity of various ifenprodil site ligands—eliprodil,(R*,R*)‐4‐hydroxy‐α‐(4‐hydroxyphenyl)‐β‐methyl‐4‐pehnyl‐1‐piperidineethanol[(±)‐CP‐101,606],cis‐3‐[4‐(4‐fluorophenyl)‐4‐hydroxy‐1‐piperidinyl]‐3,4‐dihydro‐2H‐1‐benzopyran‐4,7‐diol[(±)‐CP‐283,097], and(R*,S*)‐α‐(4‐hydroxyphenyl)‐β‐methyl‐4‐(phenylmethyl)‐1‐piperidinepropanol[(±)‐Ro 25‐6981] was very similar for inhibition of[3H]ifenprodil binding to recombinant human NR1a/NR2B and nativerat receptors, whereas allosteric inhibition of [3H]ifenprodilbinding by polyamine site ligands (spermine, spermidine, and arcaine) showedapproximately twofold lower affinity for recombinant receptors compared withnative receptors. Glutamate site ligands were less effective at modulating[3H]ifenprodil binding to recombinant NR1a/NR2B receptors comparedwith native rat receptors. The NMDA receptor‐specific[3H]ifenprodil binding conditions described were also applied to exvivo experiments to determine the receptor occupancy of ifenprodil siteligands [ifenprodil, (±)‐CP‐101,606, (±)‐CP‐283,097, and(±)‐Ro 25‐6981] given systemically.
Sensitization of Neuronal Cells to Oxidative Stress with Mutated Human α‐Synuclein
Authors: Ko Li‐wen , Mehta Nitin D. , Farrer Matthew , Easson Colin , Hussey Jennifer , Yen Samuel , Hardy John , Yen Shu‐Hui C.
Published: March 2003

Abstract: Linkage of α‐synuclein (α‐SN) mutations tofamilial Parkinson's disease (PD) and presence of α‐SN as a majorconstituent of Lawy body in both sporadic and familial PD implicate α‐SNabnormality in PD pathogenesis. Here we demonstrate that overexpression ofwild‐type or mutant α‐SN does not cause any deleterious effect on thegrowth or continued propagation of transfected human cells, but overproductionof mutant α‐SN heightens their sensitivity to menadione‐inducedoxidative injury. Such enhanced vulnerability is more pronounced in neuronaltransfectants than in their nonneuronal counterparts and is associated withincreased production of reactive oxygen species. The data suggest that mutatedα‐SN, especially with an alanine‐to‐proline substitution at residue 30,sensitizes neuronal cells to oxidative damage.
Heme Oxygenase‐1 and NADPH Cytochrome P450 Reductase Expression in Experimental Allergic Encephalomyelitis
Authors: Emerson Mitchell R. , LeVine Steven M.
Published: March 2003

Abstract: Oxidative stress is implicated in the pathogenesis ofexperimental allergic encephalomyelitis (EAE), a model for multiple sclerosis.Heme oxygenase‐1 (HO‐1) is a heat shock protein induced by oxidative stress.HO‐1 metabolizes the pro‐oxidant heme to the antioxidant biliverdin and CO.HO‐1 requires electrons, donated by NADPH cytochrome P450 reductase(henceforth, reductase), for catalytic activity. EAE was induced with apeptide of proteolipid protein in SJL mice, and the expression of HO‐1 andreductase in the hindbrain was analyzed. HO‐1 protein levels weresignificantly increased in EAE animals compared with control mice. HO‐1expression was present in ameboid macrophages, reactive microglia, andastrocytes in white matter tracks. Bergmann glia and ameboid macrophages alsowere occasionally stained in the molecular layer of the cerebellum. UnlikeHO‐1, reductase protein levels decreased with disease severity. HO‐1 andreductase were associated with each other in endoplasmic reticulum micelles,suggesting that the decrease in reductase does not interfere with itsassociation with HO‐1. In cells that express HO‐1, the association ofreductase with HO‐1 should competitively inhibit the interaction of reductasewith cytochrome P450 isozymes and thereby limit free radical production as thelatter two enzymes act cooperatively to produce superoxide. The increase inHO‐1 together with the decrease in reductase may be part of a common defensemechanism attempting to minimize tissue damage in several neurologicalconditions.
Shp‐2 Specifically Regulates Several Tyrosine‐Phosphorylated Proteins in Brain‐Derived Neurotrophic Factor Signal
Authors: Araki Toshiyuki , Yamada Masashi , Ohnishi Hiroshi , Sano Shin‐ichiro , Uetsuki Taichi , Hatanaka Hiroshi
Published: March 2003

Abstract: Brain‐derived neurotrophic factor (BDNF), a member of the neurotrophins, promotes differentiation and survival and regulates plasticity of various types of neurons. BDNF binds to TrkB, a receptor tyrosine kinase, which results in the activation of a variety of signaling molecules to exert the various functions of BDNF. Shp‐2, a Src homology 2 domain‐containing cytoplasmic tyrosine phosphatase, is involved in neurotrophin signaling in PC12 cells and cultured cerebral cortical neurons. To examine the roles of Shp‐2 in BDNF signaling in cultured rat cerebral cortical neurons, the wild‐type and phosphatase‐inactive mutant (C/S mutant) forms of Shp‐2 were ectopically expressed in cultured neurons using recombinant adenovirus vectors. We found that several proteins tyrosinephosphorylated in response to BDNF showed enhanced levels of tyrosine phosphorylation in cultured neurons infected with C/S mutant adenovirus in comparison with those infected with the wild‐type Shp‐2 adenovirus. In addition, in immunoprecipitates with anti‐Shp‐2 antibody, we also observed at least four proteins that displayed enhanced phosphorylation in response to BDNF in cultured neurons infected with the C/S mutant adenovirus. We found that the Shp‐2‐binding protein, brain immunoglobulin‐like molecule with tyrosine‐based activation motifs (BIT), was strongly tyrosine‐phosphorylated in response to BDNF in cultured neurons expressing the C/S mutant of Shp‐2. In contrast, the level of BDNF‐induced phosphorylation of mitogen‐activated protein kinase and coprecipitated proteins with anti‐Trk and Grb2 antibodies did not show any difference between neurons infected with these two types of Shp‐2. Furthermore, the survival effect of BDNF was enhanced by the wild type of Shp‐2, although it was not influenced by the C/S mutant of Shp‐2. These results indicated that in cultured cerebral cortical neurons Shp‐2 is specifically involved in the regulation of several tyrosine‐phosphorylated proteins, including BIT, in the BDNF signaling pathway. In addition, the phosphatase Shp‐2 may not influence the level of BDNF‐induced activation of mitogen‐activated protein kinase in cultured cortical neurons. Further, Shp‐2 may have potential to positively regulate BDNF‐promoting neuronal survival.
Increased Levels of Tau Protein in SH‐SY5Y Cells After Treatment with Cholinesterase Inhibitors and Nicotinic Agonists
Authors: Hellström‐Lindahl Ewa , Moore Helen , Nordberg Agneta
Published: March 2003

Abstract: Several cholinesterase inhibitors used in the treatment of Alzheimer’s disease (AD) have been shown to interact with an allosteric site on the nicotinic acetylcholine receptor (nAChR). A possible linkage between the phosphorylation state of tau, the major component of paired helical filaments found in AD brain, and stimulation of nAChRs by cholinesterase inhibitors and nicotinic agonists was investigated. Western blot analysis showed that treatment of SH‐SY5Y cells for 72 h with the cholinesterase inhibitors tacrine (10‐5M), donepezil (10‐5M), and galanthamine (10‐5M), nicotine (10‐5M), and epibatidine (10‐7M) increased tau levels as detected with Tau‐1, AT 8, and AT 270 monoclonal antibodies and binding of [3H]epibatidine. The increase in tau immunoreactivity induced by nicotine, epibatidine, and tacrine, but not the up‐regulation of nAChRs, was prevented by the antagonists d‐tubocurarine and mecamylamine. Both antagonists were synergistic with the nicotinic agonists in causing up‐regulation, but only d‐tubocurarine showed a synergistic effect with tacrine. The increased tau immunoreactivity induced by tacrine was not prevented by atropine, indicating that in terms of cholinergic receptors, tacrine modulates tau levels mainly through interactions with nAChRs and not with muscarinic receptors. Additional work is needed to determine the exact mechanism by which cholinesterase inhibitors and nicotinic agonists modulate phosphorylation and levels of tau protein.
Isolation and Functional Characterization of the Chick M5 Muscarinic Acetylcholine Receptor Gene
Authors: Creason Sharon , Tietje Kathleen M. , Nathanson Neil M.
Published: March 2003

Abstract: The chick is a widely used system for study of the actions of muscarinic acetylcholine receptors in the cardiovascular, visual, and nervous systems. We report the isolation and functional analysis of the gene encoding the chick M5 muscarinic receptor. RT‐PCR analysis indicates that the M5 receptor is expressed at low levels in embryonic chick brain and heart. When expressed in stably transfected Chinese hamster ovary cells, the M5 receptor exhibits high‐affinity binding to muscarinic antagonists and mediates robust activation of phospholipase C activity.
Insulin‐like growth factor binding protein 5 and type‐1 insulin‐like growth factor receptor are differentially regulated during apoptosis in cerebellar granule cells
Authors: Roschier Maaria , Kuusisto Erkki , Suuronen Tiina , Korhonen Pauliina , Kyrylenko Sergiy , Salminen Antero
Published: March 2003

Neuronal apoptosis is considered to play a significant role in several neuropathological conditions. However, the molecular mechanisms underlying neuronal apoptosis are poorly understood. Insulin‐like growth factor (IGF) signalling is considered to be an important regulator of neuronal differentiation, survival and apoptosis. We have examined the expression of two members of the IGF system, insulin‐like growth factor binding protein 5 (IGFBP‐5) and the type‐1 IGF receptor (IGF1R), during apoptosis of rat cerebellar granule cells (CGCs) in vitro. We describe a prominent downregulation of IGFBP‐5 mRNA and protein expression. We also show that IGF‐I increases IGFBP‐5 expression in CGCs and that the downregulation of IGFBP‐5 mRNA can be suppressed by inhibiting mRNA synthesis with actinomycin D. The expression of IGF1R mRNA showed a transient upregulation during potassium chloride (KCl) deprivation induced apoptosis, in contrast to the IGF1R protein level, which was downregulated during KCl deprivation. Our results provide insight into the expression of IGF‐related genes during neuronal apoptosis, and indicate that they mediate a protective response to the withdrawal of trophic stimulation. It seems that the expression of IGFBP‐5 and IGF1R is regulated to maximize the availability of IGF and the activity of IGF‐triggered survival signalling.
Inhibition of insulin‐like growth factor‐1 receptor and IRS‐2 signaling by ethanol in SH‐SY5Y neuroblastoma cells
Authors: Seiler Andrea E. M. , Ross Brian N. , Rubin Raphael
Published: March 2003

The effect of ethanol on insulin‐like growth factor‐1 (IGF‐I)‐mediated signal transduction and functional activation in neuronal cells was examined. In human SH‐SY5Y neuroblastoma cells, ethanol inhibited tyrosine autophosphorylation of the IGF‐I receptor. This corresponded to the inhibition of IGF‐I‐induced phosphorylation of p42/p44 mitogen‐activated/extracellular signal‐regulated protein kinase (MAPK) by ethanol. Insulin‐related substrate‐2 (IRS‐2) and focal adhesion kinase phosphorylation were reduced in the presence of ethanol, which corresponded to the prevention of lamellipodia formation (30 min). By contrast, ethanol had no effect on Shc phosphorylation when measured up to 1 h, and did not affect the association of Grb‐2 with Shc. Neurite formation at 24 h was similarly unaffected by ethanol. The data indicate that the IGF‐I receptor is a target for ethanol in SH‐SY5Y cells However, there is diversity in the sensitivity of signaling elements within the IGF‐I receptor tyrosine kinase signaling cascades to ethanol, which can be related to the inhibition of specific functional events in neuronal activation.
Relative contribution of the Na+/Ca2+ exchanger, mitochondria and endoplasmic reticulum in the regulation of cytosolic Ca2+ and catecholamine secretion of bovine adrenal chromaffin cells
Authors: Yang De‐Ming , Kao Lung‐Sen
Published: March 2003

The relative importance of mitochondria, the Na+/Ca2+ exchanger (NCX) and the endoplasmic reticulum (ER) in the regulation of the cytosolic Ca2+ concentration ([Ca2+]i) were examined in bovine chromaffin cells using fura‐2 for average [Ca2+]i and amperometry for secretory activity, which reflects the local Ca2+ concentration near the exocytotic sites. Chromaffin cells were stimulated by a high concentration of K+ when the three Ca2+ removal mechanisms were individually or simultaneously inhibited. When the mitochondrial Ca2+ uptake was inhibited, the [Ca2+]i decayed at a significantly slower rate and the secretory activity was higher than the control cells. The NCX appears to function only in the initial phase of [Ca2+]i decay and when the ER Ca2+ pump is blocked. Similarly, the ER had a significant effect on the [Ca2+]i decay and on the secretion only when the NCX was blocked. Inhibition of all three mechanisms leads to a substantial delay in [Ca2+]i recovery and an increase in the secretion. The results suggest that the three mechanisms work together in the regulation of the Ca2+ near the Ca2+ channels and exocytotic sites and therefore modulate the secretory activity. When Ca2+ diffuses away from the exocytotic sites, the mitochondrial Ca2+ uptake becomes the dominant mechanism.
N‐Acetyl‐l‐cysteine protects SHSY5Y neuroblastoma cells from oxidative stress and cell cytotoxicity: effects on β‐amyloid secretion and tau phosphorylation
Authors: Olivieri G. , Baysang G. , Meier F. , Müller‐Spahn F. , Stähelin H. B. , Brockhaus M. , Brack CH.
Published: March 2003

Redox changes within neurones are increasingly being implicated as an important causative agent in brain ageing and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). Cells have developed a number of defensive mechanisms to maintain intracellular redox homeostasis, including the glutathione (GSH) system and antioxidant enzymes. Here we examine the effects of N‐acetyl‐l‐cysteine (NAC) on β‐amyloid (Aβ) secretion and tau phosphorylation in SHSY5Y neuroblastoma cells after exposure to oxidative stress inducing/cytotoxic compounds (H2O2, UV light and toxic Aβ peptides). Aβ and tau protein are hallmark molecules in the pathology of AD while the stress factors are implicated in the aetiology of AD. The results show that H2O2, UV light, Aβ1–42 and toxic Aβ25–35, but not the inactive Aβ35–25, produce a significant induction of oxidative stress and cell cytotoxicity. The effects are reversed when cells are pre‐treated with 30 mm NAC. Cells exposed to H2O2, UV light and Aβ25–35, but not Aβ35–25, secrete significantly higher amounts of Aβ1–40 and Aβ1–42 into the culture medium. NAC pre‐treatment increased the release of Aβ1–40 compared with controls and potentiated the release of both Aβ1–40 and Aβ1–42 in Aβ25–35‐treated cells. Tau phosphorylation was markedly reduced by H2O2 and UV light but increased by Aβ25–35. NAC strongly lowered phospho‐tau levels in the presence or absence of stress treatment.
The multidrug resistance protein MRP1 mediates the release of glutathione disulfide from rat astrocytes during oxidative stress
Authors: Hirrlinger Johannes , König Jörg , Keppler Dietrich , Lindenau Jörg , Schulz Jörg B. , Dringen Ralf
Published: March 2003

The release of glutathione disulfide has been considered an important process for the maintenance of a reduced thiol redox potential in cells during oxidative stress. In cultured rat astrocytes, permanent hydrogen peroxide‐induced oxidative stress caused a rapid increase in intracellular glutathione disulfide, which was followed by the appearance of glutathione disulfide in the medium. Under these conditions, the viability of the cells was not compromised. In the presence of cyclosporin A and the quinoline‐derivative MK571, inhibitors of multidrug resistance proteins (MRP1 and MRP2), glutathione disulfide accumulated in cells and the release of glutathione disulfide from astrocytes during H2O2 stress was potently inhibited, suggesting a contribution of MRP1 or MRP2 in the release of glutathione disulfide from astrocytes. Using RT‐PCR we amplified a cDNA from astroglial RNA with a high degree of homology to MRP1 from humans and mouse. In contrast, no fragment was amplified by using primers specific for rat MRP2. In addition, the presence of MRP1 protein in astrocytes was demonstrated by its immunolocalization in cells expressing the astroglial marker protein glial fibrillary acidic protein. Our data identify rat astrocytes as a MRP1‐expressing brain cell type and demonstrate that this transporter participates in the release of glutathione disulfide from astrocytes during oxidative stress.
Synuclein‐1 is selectively up‐regulated in response to nerve growth factor treatment in PC12 cells
Authors: Stefanis Leonidas , Kholodilov Nikolai , Rideout Hardy J. , Burke Robert E. , Greene Lloyd A.
Published: March 2003

Mutations in the α‐synuclein gene have recently been identified in families with inherited Parkinson's disease and the protein product of this gene is a component of Lewy bodies, indicating that α‐synuclein is involved in Parkinson's disease pathogenesis. A role for normal α‐synuclein in synaptic function, apoptosis or plasticity responses has been suggested. We show here that in rat pheochromocytoma PC12 cells synuclein‐1, the rat homolog of human α‐synuclein, is highly and selectively up‐regulated at the mRNA and protein levels after 7 days of nerve growth factor treatment. Synuclein‐1 expression appears neither sufficient nor necessary for the neuritic sprouting that occurs within 1–2 days of nerve growth factor treatment. Rather, it likely represents a component of a late neuronal maturational response. Synuclein‐1 redistributes diffusely within the cell soma and the neuritic processes in nerve growth factor‐treated PC12 cells. Cultured neonatal rat sympathetic neurones express high levels of synuclein‐1, with a diffuse intracellular distribution, similar to neuronal PC12 cells. These results suggest that levels of synuclein‐1 may be regulated by neurotrophic factors in the nervous system and reinforce a role for α‐synuclein in plasticity‐maturational responses. In contrast, there is no correlation between synuclein expression and apoptotic death following trophic deprivation.
Differential effects of ethanol on the expression of cyclo‐oxygenase in cultured cortical astrocytes and neurons
Authors: Luo Jia , Lindström Christine L. B. , Donahue Arthur , Miller Michael W.
Published: March 2003

The developing central nervous system is a primary target of ethanol toxicity. The teratogenic effect of ethanol may result from its action on prostaglandins. Prostaglandins are generated through the release of arachidonic acid (AA) by the action of cytosolic phospholipase A2 (cPLA2) on membrane‐bound phospholipids and the catalytic conversion of AA to prostaglandin E2 (PGE2) by cyclo‐oxygenase (COX). COX is expressed in two isoforms, constitutive COX1 and inducible COX2. Cultured astrocytes and neurons from immature cerebral cortex were used as in vitro models to investigate the effect of ethanol on PGE2 synthesis. In both cell types, neither the activity nor the expression of cPLA2 was affected by ethanol. PGE2 was synthesized by astrocytes and neurons. Ethanol (200–400 mg/dL for 24 h) significantly increased PGE2 production in both cell types and the ethanol‐induced increase in PGE2 accumulation in astrocytes was significantly greater than in neurons. These increases resulted from the effects of ethanol on COX. Overall COX activity was up‐regulated by ethanol in astrocytes and neurons, and indomethacin, a nonselective blocker for COX, eliminated the ethanol‐induced increases of COX activity in both cell types. Increased COX activity in astrocytes resulted from an increase in COX2 expression. NS‐398, a selective COX2 blocker, completely inhibited ethanol‐induced alterations in COX activity. In neurons, however, ethanol had a direct effect on COX activity in the absence of a change in COX expression. NS‐398 only partially blocked ethanol‐induced increases in neuronal COX activity. Thus, astrocytes are a primary target of ethanol and ethanol‐induced increases in glial PGE2 synthesis are mediated by COX, principally COX2. Ethanol toxicity may be mediated through PGE2 in immature cortical cells.
Inhibition of stress‐ or anxiogenic‐drug‐induced increases in dopamine release in the rat prefrontal cortex by long‐term treatment with antidepressant drugs
Authors: Dazzi Laura , Spiga Francesca , Pira Luigi , Ladu Stefania , Vacca Giada , Rivano Antonella , Jentsch J. David , Biggio Giovanni
Published: March 2003

The effects of long‐term treatment with imipramine or mirtazapine, two antidepressant drugs with different mechanisms of action, on the response of cortical dopaminergic neurons to foot‐shock stress or to the anxiogenic drug FG7142 were evaluated in freely moving rats. As expected, foot shock induced a marked increase (+ 90%) in the extracellular concentration of dopamine in the prefrontal cortex of control rats. Chronic treatment with imipramine or mirtazapine inhibited or prevented, respectively, the effect of foot‐shock stress on cortical dopamine output. Whereas acute administration of the anxiogenic drug FG7142 induced a significant increase (+ 60%) in cortical dopamine output in control rats, chronic treatment with imipramine or mirtazapine completely inhibited this effect. In contrast, the administration of a single dose of either antidepressant 40 min before foot shock, had no effect on the response of the cortical dopaminergic innervation to stress. These results show that long‐term treatment with imipramine or mirtazapine inhibits the neurochemical changes elicited by stress or an anxiogenic drug with an efficacy similar to that of acute treatment with benzodiazepines. Given that episodes of anxiety or depression are often preceded by stressful events, modulation by antidepressants of the dopaminergic response to stress might be related to the anxiolytic and antidepressant effects of these drugs.
Nitrogen shuttling between neurons and glial cells during glutamate synthesis
Authors: Lieth Erich , LaNoue Kathryn F. , Berkich Deborah A. , Xu Baiyang , Ratz Michael , Taylor Charles , Hutson Susan M.
Published: March 2003

The relationship between neuronal glutamate turnover, the glutamate/glutamine cycle and de novo glutamate synthesis was examined using two different model systems, freshly dissected rat retinas ex vivo and in vivo perfused rat brains. In the ex vivo rat retina, dual kinetic control of de novo glutamate synthesis by pyruvate carboxylation and transamination of α‐ketoglutarate to glutamate was demonstrated. Rate limitation at the transaminase step is likely imposed by the limited supply of amino acids which provide the α‐amino group to glutamate. Measurements of synthesis of 14C‐glutamate and of 14C‐glutamine from H14CO3 have shown that 14C‐amino acid synthesis increased 70% by raising medium pyruvate from 0.2 to 5 mm. The specific radioactivity of 14C‐glutamine indicated that ∼30% of glutamine was derived from 14CO2 fixation. Using gabapentin, an inhibitor of the cytosolic branched‐chain aminotransferase, synthesis of 14C‐glutamate and 14C‐glutamine from H14CO3− was inhibited by 31%. These results suggest that transamination of α‐ketoglutarate to glutamate in Müller cells is slow, the supply of branched‐chain amino acids may limit flux, and that branched‐chain amino acids are an obligatory source of the nitrogen required for optimal rates of de novo glutamate synthesis. Kinetic analysis suggests that the glutamate/glutamine cycle accounts for 15% of total neuronal glutamate turnover in the ex vivo retina. To examine the contribution of the glutamate/glutamine cycle to glutamate turnover in the whole brain in vivo, rats were infused intravenously with H14CO3−. 14C‐metabolites in brain extracts were measured to determine net incorporation of 14CO2 and specific radioactivity of glutamate and glutamine. The results indicate that 23% of glutamine in the brain in vivo is derived from 14CO2 fixation. Using published values for whole brain neuronal glutamate turnover, we calculated that the glutamate/glutamine cycle accounts for ∼60% of total neuronal turnover. Finally, differences between glutamine/glutamate cycle rates in these two model systems suggest that the cycle is closely linked to neuronal activity.
Signaling pathways in the induction of c‐met receptor expression by its ligand scatter factor/hepatocyte growth factor in human glioblastoma
Authors: Abounader R. , Ranganathan S. , Kim B. Y. S. , Nichols C. , Laterra J.
Published: March 2003

Scatter factor/hepatocyte growth factor (SF/HGF) and its tyrosine kinase receptor c‐met are developmentally expressed, neuroprotective, and tumorigenic within the CNS. In the present study SF/HGF is shown to induce the expression of c‐met in two human glioblastoma cell lines, U‐373 MG and T98G, and the signaling pathways involved in this induction are dissected. SF/HGF activated mitogen‐activated protein kinase (MAPK) and inhibition of either Ras or MAPK‐kinase completely inhibited SF/HGF‐mediated c‐met induction. Inhibition of phospholipase‐C (PLC) did not affect c‐met induction in either cell line. Inhibition of phosphoinositide 3‐kinase (PI3‐kinase) substantially reduced c‐met induction by SF/HGF in T98G cells but had no effect in U‐373 MG cells. Protein kinase C (PKC) inhibition reduced c‐met induction in T98G cells but not in U‐373 MG cells. SF/HGF induced the expression of c‐fos and c‐jun mRNA and increased the levels of AP‐1 transcription factor in both cells lines as determined by AP‐1‐luciferase reporter expression. Transfection of either cell line with TAM‐67, a dominant negative for the jun transactivation domain, completely inhibited AP‐1 and c‐met induction by SF/HGF. These results support a model of c‐met induction by SF/HGF in human glioma cells that uniformly involves Ras, MAPK, and AP‐1 and additionally involves PI3‐kinase and PKC in some cell lines.