bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin‐Like Growth Factor I Prevents the Development of Sensitivity to Kainate Neurotoxicity in Cerebellar Granule Cells
Authors: Leski Michael L. , Valentine Stacey L. , Baer Jesse D. , Coyle Joseph T.
Published: March 2003

Abstract: This study reports that insulin‐like growth factor I
(IGF‐I) prevents cerebellar granule cells from developing sensitivity to
kainate neurotoxicity. Sensitivity to kainate neurotoxicity normally develops
5‐6 days after switching cultures to a serum‐free medium containing 25
mM K+. Addition of either IGF‐I or insulin to the
serum‐free medium at the time of the switch prevented the development of
sensitivity to kainate, whereas brain‐derived neurotrophic factor,
neurotrophin‐3, neurotrophin‐4, and nerve growth factor did not. The
dose‐response curves indicated IGF‐I was more potent than insulin, favoring
the assignment of the former as the physiological protective agent. The
phosphatidylinositol 3‐kinase (PI 3‐K) inhibitors wortmannin (10‐100
nM) and LY 294002 (0.3‐1 μM) abolished the protection
afforded by IGF‐I. The p70 S6 kinase (p70S6k) inhibitor rapamycin
(5‐50 nM) also abolished the protection afforded by IGF‐I. The
activities of both enzymes decreased in cultures switched to serum‐free medium
but increased when IGF‐I was included; wortmannin (100 nM) lowered
the activity of PI 3‐K from 2 to 5 days after medium switch, whereas rapamycin
(50 nM) prevented the increase observed for p70S6k
activity over the same interval. The mitogen‐activated protein kinase kinase
inhibitor U 0126 and the mitogen‐activated protein kinase inhibitor SB 203580
did not abolish IGF‐I protection. Kainate neurotoxicity was not prevented by
Joro spider toxin; therefore, the development of kainate neurotoxicity could
not be explained by the formation of calcium‐permeable
α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptors. These
results indicate that IGF‐I functions through a signal transduction pathway
involving PI 3‐K and p70S6k to prevent the development of sensitivity to kainate neurotoxicity in cerebellar granule cells.
Lysophosphatidic Acid Inhibits Ca2+ Signaling in Response to Epidermal Growth Factor Receptor Stimulation in Human Astrocytoma Cells by a Mechanism Involving Phospholipase Cγ and a Gαi Protein
Authors: Hernández Marita , Barrero María José , Crespo Mariano Sánchez , Nieto María Luisa
Published: March 2003

Abstract: The effect of the lysophospholipid mediators
lysophosphatidic acid (LPA) and sphingosine 1‐phosphate and the polypeptide
growth factor epidermal growth factor (EGF) on the human astrocytoma cell line
1321N1 was assessed. These agonists produced a rapid and transient increase of
the intracellular Ca2+ concentration. When LPA was perfused before
addition of EGF, the EGF‐dependent Ca2+ transient was abrogated,
whereas this was not observed when EGF preceded LPA addition. This inhibitory
effect was not found for other EGF‐mediated responses, e.g., activation of the
mitogen‐activated protein kinase cascade and cell proliferation, thus pointing
to the existence of cross‐talk between LPA and EGF for only a branch of
EGF‐induced responses. As 1321N1 cells expressed mRNA encoding the LPA
receptors endothelial differentiation gene (Edg)‐2, Edg‐4, and Edg‐7 and as
sphingosine 1‐phosphate did not interfere with LPA signaling, Edg‐2, Edg‐4,
and/or Edg‐7 could be considered as the LPA receptors mediating the
aforementioned cross‐talk. Attempts to address the biochemical mechanism
involved in the cross‐talk between the receptors were conducted by the
immunoprecipitation approach using antibodies reacting with the EGF receptor
(EGFR), phosphotyrosine, phospholipase Cγ (PLCγ)‐1, and
Gαi protein. LPA was found to induce coupling of PLCγ‐1
to the EGFR by a mechanism involving a Gαi protein, in the
absence of tyrosine phosphorylation of both PLCγ and the EGFR. These
data show a cross‐talk between LPA and EGF limited to a branch of
EGFR‐mediated signaling, which may be explained by a LPA‐induced,
Gαi‐protein‐mediated translocation of PLCγ‐1 to EGFR in the absence of detectable tyrosine phosphorylation of both proteins.
D‐Glucose Prevents Glutathione Oxidation and Mitochondrial Damage After Glutamate Receptor Stimulation in Rat Cortical Primary Neurons
Authors: Delgado‐Esteban María , Almeida Angeles , Bolaños Juan P.
Published: March 2003

Abstract: The possible neuroprotective effect of D‐glucose against
glutamate‐mediated neurotoxicity was studied in rat cortical neurons in
primary culture. Brief (5‐min) exposure of neurons to glutamate (100
μM) increased delayed (24‐h) necrosis and apoptosis by 3‐ and
1.8‐fold, respectively. Glutamate‐mediated neurotoxicity was accompanied by a
D‐(‐)‐2‐amino‐5‐phosphonopentanoate (100 μM) and
Nω‐nitro‐L‐arginine methyl ester (1
mM)‐inhibitable, time‐dependent ATP depletion (55% at 24 h),
confirming the involvement of NMDA receptor stimulation followed by nitric
oxide synthesis in this process. Furthermore, the presence of D‐glucose (20
mM), but not its inactive enantiomer, L‐glucose, fully prevented
glutamate‐mediated delayed ATP depletion, necrosis, and apoptosis. Succinate‐
cytochrome c reductase activity, but not the activities of
NADH‐coenzyme Q1 reductase or cytochrome c oxidase, was inhibited by 32% by glutamate treatment, an effect that was abolished by incubation with D‐glucose. Lactate accumulation in the culture medium was unmodified by any of these treatments, ruling out the possible involvement of the glycolysis pathway in either glutamate neurotoxicity or D‐glucose neuroprotection. In contrast, D‐glucose, but not L‐glucose, abolished glutamate‐mediated glutathione oxidation and NADPH depletion. Our results suggest that NADPH production from D‐glucose accounts for glutathione regeneration and protection from mitochondrial dysfunction. This supports the notion that the activity of the pentose phosphate pathway may be an important factor in protecting neurons against glutamate neurotoxicity.
Somatodendritic Localization of Translin, a Component of the Translin/Trax RNA Binding Complex
Authors: Finkenstadt Patricia M. , Kang Wha‐Sun , Jeon Mihee , Taira Eiichi , Tang Weizhu , Baraban Jay M.
Published: March 2003

Abstract: Recent studies implicating dendritic protein synthesis in
synaptic plasticity have focused attention on identifying components of the
molecular machinery involved in processing dendritic RNA. Although Translin
was originally identified as a protein capable of binding single‐stranded DNA,
subsequent studies have demonstrated that it also binds RNAin vitro. Because previous studies indicated that Translin‐containing RNA/single‐stranded DNA binding complexes are highly enriched in brain, we and others have proposed that it may be involved in dendritic RNA processing. To assess this possibility, we have conducted studies aimed at defining the localization of Translin and its partner protein, Trax, in brain. In situ hybridization studies demonstrated that both Translin and Trax are expressed in neurons with prominent staining apparent in cerebellar Purkinje cells and neuronal layers of the hippocampus. Subcellular fractionation studies demonstrated that both Translin and Trax are highly enriched in the cytoplasmic fraction compared with nuclear extracts. Furthermore, immunohistochemical studies with Translin antibodies revealed prominent staining in Purkinje neuron cell bodies that extends into proximal and distal dendrites. A similar pattern of somatodendritic localization was observed in hippocampal and neocortical pyramidal neurons. These findings demonstrate that Translin is expressed in neuronal dendrites and therefore support the hypothesis that the Translin/Trax complex may be involved in dendritic RNA processing.
Target‐Specific Glutamatergic Regulation of Dopamine Neurons in the Ventral Tegmental Area
Authors: Takahata Ryuichi , Moghaddam Bita
Published: March 2003

Abstract: Dopamine (DA) neurons in the ventral tegmental area (VTA) are thought to play a critical role in affective, motivational, and cognitive functioning. There are fundamental target‐specific differences in the functional characteristics of subsets of these neurons. For example, DA afferents to the prefrontal cortex (PFC) have a higher firing and transmitter turnover rate and are more responsive to some pharmacological and environmental stimuli than DA projections to the nucleus accumbens (NAc). These functional differences may be attributed in part to differences in tonic regulation by glutamate. The present study provides evidence for this mechanism: In freely moving animals, blockade of basal glutamatergic activity in the VTA by the selective α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA)/kainate antagonist LY293558 produced an increase in DA release in the NAc while significantly decreasing DA release in the PFC. These data support an AMPA receptor‐mediated tonic inhibitory regulation of mesoaccumbens neurons and a tonic excitatory regulation of mesoprefrontal DA neurons. This differential regulation may result in target‐specific effects on the basal output of DA neurons and on the regulatory influence of voltage‐gated NMDA receptors in response to phasic activation by behaviorally relevant stimuli.
Blood—Brain Barrier Is Involved in the Efflux Transport of a Neuroactive Steroid, Dehydroepiandrosterone Sulfate, via Organic Anion Transporting Polypeptide 2
Authors: Asaba Hiroshi , Hosoya Ken‐ichi , Takanaga Hitomi , Ohtsuki Sumio , Tamura Etsuko , Takizawa Takuya , Terasaki Tetsuya
Published: March 2003

Abstract: We have investigated the transport characteristics of
dehydroepiandrosterone sulfate (DHEAS), a neuroactive steroid, at the
blood—brain barrier (BBB) in a series of functional in vivo and in vitro
studies. The apparent BBB efflux rate constant of [3H]DHEAS
evaluated by the brain efflux index method was 2.68 × 10‐2
min‐1. DHEAS efflux transport was a saturable process with a
Michaelis constant (Km) of 32.6 μM.
Significant amounts of [3H]DHEAS were determined in the jugular
venous plasma by HPLC, providing direct evidence that most of the DHEAS is
transported in intact form from brain to the circulating blood across the BBB.
This efflux transport of [3H]DHEAS was significantly inhibited by
common rat organic anion‐transporting polypeptide (oatp) substrates such as
taurocholate, cholate, sulfobromophthalein, and estrone‐3‐sulfate. Moreover,
the apparent efflux clearance of [3H]DHEAS across the BBB (118
μl/min‐g of brain) was 10.4‐fold greater than its influx clearance
estimated by the in situ brain perfusion technique (11.4 μl/min‐g of
brain), suggesting that DHEAS is predominantly transported from the brain to
blood across the BBB. In cellular uptake studies using a conditionally
immortalized mouse brain capillary endothelial cell line (TM‐BBB4),
[3H]DHEAS uptake by TM‐BBB4 cells exhibited a concentration
dependence with a Km of 34.4 μM and was
significantly inhibited by the oatp2‐specific substrate digoxin. Conversely,
[3H]digoxin uptake by TM‐BBB4 cells was significantly inhibited by
DHEAS. Moreover, the net uptake of [3H]DHEAS at 30 min was significantly increased under ATP‐depleted conditions, suggesting that an energy‐dependent efflux process may also be involved in TM‐BBB4. RT‐PCR and sequence analysis suggest that an oatp2 is expressed in TM‐BBB4 cells. In conclusion, DHEAS efflux transport takes place across the BBB, and studies involving in vitro DHEAS uptake and RT‐PCR suggest that there is oatp2‐mediated DHEAS transport at the BBB.
Involvement of α7 Nicotinic Acetylcholine Receptors in Activation of Tyrosine Hydroxylase and Dopamine β‐Hydroxylase Gene Expression in PC12 Cells
Authors: Gueorguiev Volodia D. , Zeman Richard J. , Meyer Edwin M. , Sabban Esther L.
Published: March 2003

Abstract: Nicotine treatment increases intracellular free
Ca2+ concentration [Ca2+]i, stimulates
catecholamine release, and elevates gene expression for the catecholamine
biosynthetic enzymes tyrosine hydroxylase (TH) and dopamine β‐hydroxylase
(DBH). However, the type of nicotinic acetylcholine receptors (nAChRs)
mediating these events is unclear. The nAChR receptor antagonists
α‐bungarotoxin (αBTX) and methyllycaconitine greatly reduced the
nicotine‐triggered initial transient rise in [Ca2+]i and
prevented the second prolonged elevation of [Ca2+]i,
suggesting the involvement of α7 nAChRs. Two specific α7 nicotinic
agonists, 3‐(2,4‐dimethoxybenzilidene)anabaseine (DMXB) and
E,E‐3‐(cinnamylidene)anabaseine (3‐CA), were found to elicit a small,
delayed increase in [Ca2+]i with kinetics and magnitude
similar to the second elevation observed with nicotine. This increase was
inhibited by the inositol trisphosphate receptor antagonist xestospongin C.
Exposure to 3‐CA or DMXB for 6 or 24 h elevated TH and DBH mRNA levels two‐ to
fourfold over control levels. These agonists were more effective than nicotine
alone in increasing TH and DBH gene expression and significantly elevated
[Ca2+]i for up to 6 h. The increase in
[Ca2+]i or the elevation in TH mRNA by 3‐CA was completely inhibited by αBTX. This study, for the first time, implicates stimulation of α7 nAChRs in the activation of TH and DBH gene expression.
Interleukin‐1β Induces Cyclooxygenase‐2 and Prostaglandin E2 Synthesis in Human Neuroblastoma Cells
Authors: Fiebich Bernd L. , Mueksch Barbara , Boehringer Martina , Hüll Michael
Published: March 2003

Abstract: Prostaglandins (PGs), which are generated by the enzymatic
activity of cyclooxygenase (COX)‐1 and ‐2, modulate several functions in the
CNS such as the generation of fever, the sleep/wake cycle, and the perception
of pain. Moreover, the neuronal induction of COX‐2 has been linked to
neuroinflammatory aspects of Alzheimer's disease (AD). The regulation of COX
expression in neuronal cells is only partly understood and has been mainly
linked to synaptic activity. In pathophysiological situations, however,
cytokines may be potent stimulators of neuronal COX expression. Here we show
that interleukin (IL)‐1β induces COX‐2 mRNA and protein synthesis and the
release of PGE2 in the human neuroblastoma cell line SK‐N‐SH. We further demonstrate that both a free radical scavenger and an inhibitor of p38 mitogen‐activated protein kinase (MAPK) reduce IL‐1β‐induced synthesis of COX‐2. IL‐1β induces p38 MAPK phosphorylation and activation of the nuclear factor‐κB independently from each other. Our data suggest that IL‐1β‐induced COX‐2 expression in SK‐N‐SH cells is regulated by different mechanisms, presumably involving mRNA transcription and mRNA stability. The ability of p38 MAPK to augment COX‐2 expression in human neuroblastoma cells, as shown here, suggests that p38 MAPK may be involved in neuronal expression of COX‐2 in AD.
Age‐Dependent Differential Responses of Monoaminergic Systems to High Doses of Methamphetamine
Authors: Kokoshka Jerry M. , Fleckenstein Annette E. , Wilkins Diana G. , Hanson Glen R.
Published: March 2003

Abstract: Abuse of methamphetamine (METH) by adolescents is a major public health issue in the U.S.A. Because of the neurotoxic potential of METH, we examined the response of CNS monoaminergic systems in young (adolescent) animals [postnatal day (PND) 40] to high‐dose treatments (10 mg/kg, four injections, 2‐h intervals) of this drug and contrasted these effects to those seen in older (young adult) rats (PND 90). Consistent with previous reports, we observed that PND 40 animals did not manifest the long‐term (7‐day) deficits in extrapyramidal dopamine (DA) parameters observed in PND 90 rats. In contrast, METH‐induced rapid (1‐h) reduction in the activity of striatal DA transporters occurred in both age groups. In addition, both persistent (7‐day) and rapid (1‐h) deficits in serotonergic systems (measured as reductions in tryptophan hydroxylase activity) were observed in PND 40 and 90 rats. Age‐related differences in METH‐induced hyperthermia did not appear to be a principal cause for our observations; however, age‐dependent pharmacokinetics of this drug might have contributed to the differential METH monoaminergic responses by PND 40 and 90 animals.
Haloperidol‐Induced Neuronal Apoptosis
Authors: Noh Jai Sung , Kang Hyo Jung , Kim Eun Young , Sohn Seonghyang , Chung Young Ki , Kim Seung U. , Gwag Byoung J.
Published: March 2003

Abstract: We examined patterns and mechanisms of cell death inducedby haloperidol. Cortical cell cultures exposed to 10‐100 μMhaloperidol for 24 h underwent neuronal death without injuring glia. Thedegenerating neurons showed hallmarks of apoptosis, featuring cell bodyshrinkage, nuclear chromatin condensation and aggregation, nuclear membranedisintegration with intact plasma membrane, and prominent internucleosomal DNAfragmentation. Neither glutamate antagonists nor antioxidants prevented thehaloperidol‐induced neuronal apoptosis. The c‐Jun‐NH2‐terminalprotein kinase and p38 mitogen‐activated protein kinase were activated within1 h and were sustained over the next 3 h following exposure of corticalneurons to 30 μM haloperidol. Haloperidol‐induced neuronalapoptosis was partially attenuated by 10‐30 μM PD169316, aselective inhibitor of p38 mitogen‐activated protein kinase. Inclusion of 1μg/ml cycloheximide, a protein synthesis inhibitor, or 100 ng/ml insulinprevented activation of both kinases and subsequent neuronal death. Thepresent study demonstrates that cortical neurons exposed to haloperidolundergo apoptosis depending on activation of p38 mitogen‐activated proteinkinase and c‐Jun‐NH2‐terminal protein kinase sensitive tocycloheximide and insulin.
Loss of Cannabinoid‐Stimulated Guanosine 5′‐O‐(3‐[35S]Thiotriphosphate) Binding Without Receptor Down‐Regulation in Brain Regions of Anandamide‐Tolerant Rats
Authors: Rubino T. , Viganò D. , Costa B. , Colleoni M. , Parolaro D.
Published: March 2003

Abstract: The endogenous cannabinoid anandamide has been reported toproduce well‐defined behavioral tolerance, but studies on the possiblemechanisms underlying this process are few and often contradictory. Thepresent study was designed to survey the cellular events involved inanandamide tolerance, in terms of the effects on receptor number, couplingwith G proteins, and activation of the cyclic AMP (cAMP) cascade. Chronictreatment of rats with anandamide (20 mg/kg i.p. for 15 days) resulted inbehavioral tolerance without any change in cannabinoid receptor binding in thebrain regions studied (striatum, cortex, hippocampus, and cerebellum),suggesting that receptor down‐regulation was not involved in the developmentof anandamide behavioral tolerance. In contrast, prolonged exposure toanandamide significantly reduced agonist‐stimulated guanosine5′‐O‐(3‐[35S]thiotriphosphate) binding in the sameareas, with losses of >50%, suggesting that receptor desensitization may bepart of the molecular mechanism underlying this tolerance. Finally, concerningthe cAMP cascade—the most well‐known intracellular signaling pathwaysactivated by CB1 receptors—in the brain regions from ratstolerant to anandamide, we found no alteration in cAMP levels or in proteinkinase A activity. We propose that anandamide, unlikeΔ9‐tetrahydrocannabinol and other cannabinoids, does notalter the receptor system at multiple levels but that desensitization of theCB1 receptor might account for behavioral tolerance to thedrug.
The Role of Prion Peptide Structure and Aggregation in Toxicity and Membrane Binding
Authors: Rymer Dawn L. , Good Theresa A.
Published: March 2003

Abstract: Prion diseases are neurodegenerative disorders associatedwith a conformational change in the normal cellular isoform of the prionprotein, PrPC, to an abnormal scrapie isoform, PrPSC.Unlike the α‐helical PrPC, the protease‐resistant core ofPrPSC is predominantly β‐sheet and possesses a tendency topolymerize into amyloid fibrils. We performed experiments with two synthetichuman prion peptides, PrP(106‐126) and PrP(127‐147), to determine how peptidestructure affects neurotoxicity and protein‐membrane interactions. Peptidesolutions possessing β‐sheet and amyloid structures were neurotoxic toPC12 cells in vitro and bound with measurable affinities to cholesterol‐richphospholipid membranes at ambient conditions, but peptide solutions lackingstable β‐sheet structures and amyloid content were nontoxic and possessedless than one tenth of the binding affinities of the amyloid‐containingpeptides. Regardless of structure, the peptide binding affinities tocholesterol‐depleted membranes were greatly reduced. These results suggestthat the β‐sheet and amyloid structures of the prion peptides give riseto their toxicity and membrane binding affinities and that membrane bindingaffinity, especially in cholesterol‐rich environments, may be related totoxicity. Our results may have significance in understanding the role of thefibrillogenic cerebral deposits associated with some of the prion diseases inneurodegeneration and may have implications for other amyloidoses.
Activation of Nuclear Factor‐κB in C6 Rat Glioma Cells After Transfection with Glia Maturation Factor
Authors: Lim Ramon , Zaheer Asgar , Yorek Mark A. , Darby Christine J. , Oberley Larry W.
Published: March 2003

Abstract: The 17‐kDa endogenous brain protein glia maturation factor (GMF) was transfected into C6 rat glioma cells using a replication‐defective human adenovirus vector. The cells overexpressed GMF but did not secrete the protein into the medium. Transfection with GMF led to the activation of the transcription factor nuclear factor‐κB (NF‐κB), as evidenced by electrophoretic mobility shift assay of the nuclear extract, using a double‐stranded oligonucleotide probe containing the consensus binding sequence for NF‐κB. The specificity of binding was demonstrated by competition with unlabeled probe and by the nonbinding of the mutant probe. Binding was detectable as early as 3 h after transfection, peaked at 6 and 12 h, and gradually declined thereafter. The observed NF‐κB activation was reduced by cotransfection with catalase and by the presence of high concentrations of pyruvate in the medium, suggesting the involvement of H2O2. The p38 mitogen‐activated protein kinase inhibitor SB‐203580 also suppressed the GMF‐activated NF‐κB, suggesting the involvement of the p38 signal transduction cascade. On the other hand, the phorbol ester phorbol 12‐myristate 13‐acetate activated NF‐κB whether or not GMF was overexpressed. Along with NF‐κB activation was an enhanced expression of superoxide dismutase (SOD), which was suppressed if NF‐κB nuclear translocation was blocked by its specific decoy DNA, implicating NF‐κB as an upstream mediator of this anti‐oxidant enzyme. The p38 inhibitor SB‐203580 also blocked the GMF‐activated SOD. As NF‐κB and SOD are both pro‐survival signals, the results suggest a cytoprotective role for endogenous GMF in glial cells.
Differences in central noradrenergic and behavioural responses of Maudsley non‐reactive and Maudsley reactive inbred rats on exposure to an aversive novel environment
Authors: McQuade Richard , Stanford S. Clare
Published: March 2003

The present experiments compared the noradrenaline and behavioural responses of inbred Maudsley reactive (MR) and non‐reactive (MNRA) rats when they are exposed to the light or dark arena of a light/dark shuttle‐box. Behavioural scores confirmed that both strains of rats perceived the light arena to be more aversive than the dark one. Using in vivo microdialysis, exposure to the light, but not the dark, arena was found to increase noradrenaline efflux in both the frontal cortex and the hypothalamus of MNRA and MR rats. However, whereas the increase in the frontal cortex of both strains and the hypothalamus of MR rats was transient, the hypothalamic response in MNRA rats was maintained throughout exposure to the test zone. Strain differences in activity/visit and time/visit were evident but it was not possible to discern whether this could be attributed to the strain difference in the hypothalamic noradrenaline response. Nevertheless, it remains possible that, by comparison with MR rats, the prolonged noradrenaline response in the hypothalamus of MNRA rats could contribute to their well‐documented, greater resistance to aversive environmental stimuli.
Expression and localization of the CYP2B subfamily predominantly in neurones of rat brain
Authors: Rosenbrock Holger , Hagemeyer Christoph E. , Ditter Margarethe , Knoth Rolf , Volk Benedikt
Published: March 2003

Despite the very small amounts of cytochrome P450 (P450, CYP) enzymes expressed in different areas and cell populations of the brain as compared with the liver, there is significant evidence for their specific involvement in brain development, function and plasticity. Nevertheless, the current discussion about occurrence and importance of cerebral cytochrome P450s is determined by inconsistent interpretations of their function in general and with respect to single isoforms. Continuing a series of publications about brain P450 isoforms, we now present evidence for the constitutive expression of CYP2B1 and CYP2B2 mRNAs in rat brain. Immunocytochemical and non‐radioactive in situ hybridization studies revealed the same expression pattern throughout the brain predominantly in neuronal populations, but to some extent in astrocytes of corpus callosum and olfactory bulb. The well known testosterone‐metabolizing capacity and the presence of CYP2B isoforms shown in steroid hormone‐sensitive areas and neurones (e.g. hippocampus) clarify the significance of isoforms like CYP2B1 and CYP2B2 for impairment of steroid hormone actions by P450 inducing environmental substances. We argue that cerebral P450 isoforms which are induced by xenobiotics and are able to metabolize these as well as endogenous substrates help us to understand fundamental aspects of brain's functioning.
Effects of neuropathic and non‐neuropathic isomers of tricresyl phosphate and their microsomal activation on the production of axon‐like processes by differentiating mouse N2a neuroblastoma cells
Authors: Fowler Maxine J. , Flaskos John , McLean W. Graham , Hargreaves Alan J.
Published: March 2003

The aim of this work was to investigate the sublethal neuropathic effects of tricresyl phosphate (TCP: mixed isomers), triorthocresyl phosphate (TOCP) and triparacresyl phosphate (TPCP) on differentiating mouse N2a neuroblastoma cells. This was achieved by a combination of measurements of cell viability, axon outgrowth and the levels of cytoskeletal proteins detectable on western blots of extracts from cells induced to differentiate in the presence and absence of the compounds. In a time‐course experiment TCP inhibited the outgrowth of axon‐like processes following exposure times of 24 h or longer. Dose–response experiments indicated that TCP and TOCP exhibited similar sustained levels of toxicity following both 24 and 48 h exposure, with no significant difference between their respective IC50 values. By contrast, TPCP demonstrated a transient effect on the outgrowth of axon‐like processes, which was detectable after 24 but not 48 h of exposure. Isomer‐specific patterns of toxicity were also evident at earlier time‐points, with only the ortho isomer showing significant levels of inhibition of axon outgrowth following 4–8 h exposure. Probing of western blots with antibodies against cytoskeletal proteins indicated that the inhibition of axon outgrowth by these compounds was associated with a sustained reduction in the levels of phosphorylated neurofilament heavy chain. The inhibitory effect on axon outgrowth of TOCP but not TPCP was enhanced in the presence of a microsomal activation system. Since TOCP is the most neuropathic of the isomers of TCP in vivo, differentiating N2a cells provide a useful cellular system for mechanistic studies of the neurodegenerative effects of this organophosphate.
The solubility of α‐synuclein in multiple system atrophy differs from that of dementia with Lewy bodies and Parkinson's disease
Authors: Campbell Bruce C. V. , McLean Catriona A. , Culvenor Janetta G. , Gai Wei Ping , Blumbergs Peter C. , Jäkälä Pekka , Beyreuther Konrad , Masters Colin L. , Li Qiao‐Xin
Published: March 2003

Intracellular inclusions containing α‐synuclein (αSN) are pathognomonic features of several neurodegenerative disorders. Inclusions occur in oligodendrocytes in multiple system atrophy (MSA) and in neurons in dementia with Lewy bodies (DLB) and Parkinson's disease (PD). In order to identify disease‐associated changes of αSN, this study compared the levels, solubility and molecular weight species of αSN in brain homogenates from MSA, DLB, PD and normal aged controls. In DLB and PD, substantial amounts of detergent‐soluble and detergent‐insoluble αSN were detected compared with controls in grey matter homogenate. Compared with controls, MSA cases had significantly higher levels of αSN in the detergent‐soluble fraction of brain samples from pons and white matter but detergent‐insoluble αSN was not detected. There was an inverse correlation between buffered saline‐soluble and detergent‐soluble levels of αSN in individual MSA cases suggesting a transition towards insolubility in disease. The differences in solubility of αSN between grey and white matter in disease may result from different processing of αSN in neurons compared with oligodendrocytes. Highly insoluble αSN is not involved in the pathogenesis of MSA. It is therefore possible that buffered saline‐soluble or detergent‐soluble forms of αSN are involved in the pathogenesis of other αSN‐related diseases.
Ser‐59 is the major phosphorylation site in αB‐crystallin accumulated in the brains of patients with Alexander's disease
Authors: Kato Kanefusa , Inaguma Yutaka , Ito Hidenori , Iida Kayo , Iwamoto Ikuko , Kamei Keiko , Ochi Nobuhiko , Ohta Hideomi , Kishikawa Masao
Published: March 2003

The phosphorylation state of αB‐crystallin accumulated in the brains of two patients with Alexander's disease (one infantile and one juvenile type) was determined by means of SDS‐PAGE or isoelectric focusing of soluble and insoluble fractions of brain extracts and subsequent western blot analysis with specific antibodies against αB‐crystallin and each of three phosphorylated serine residues. The level of mammalian small heat shock protein of 25–28 kDa (Hsp27) in the same fraction was also estimated by western blot analysis. The majority of αB‐crystallin was detected in the insoluble fraction of brain homogenates and phosphorylation was preferentially observed at Ser‐59 in both cases. A significant level of phosphorylation at Ser‐45 but not Ser‐19 was also detected. Hsp27 was found at considerable levels in the insoluble fractions. αB‐crystallin and phosphorylated forms were detected in the cerebrospinal fluid of patient with the juvenile type. αB‐crystallin and phosphorylated forms were also detectable at considerable levels in the insoluble fraction of brain homogenates from patients with Alzheimer's disease and aged controls. The phosphorylation site was mostly at Ser‐59 in all cases. Immunohistochemically, αB‐crystallin was stained in Rosenthal fibers in brains of patients with Alexander's disease and their peripheral portions were immunostained with antibodies recognizing phosphorylated Ser‐59. These results indicate that the major phosphorylation site in αB‐crystallin in brains of patients with Alexander's disease or Alzheimer's disease as well as in aged controls is Ser‐59.
Studies on the effects of lactate transport inhibition, pyruvate, glucose and glutamine on amino acid, lactate and glucose release from the ischemic rat cerebral cortex
Authors: Phillis J. W. , Ren J. , O'regan M. H.
Published: March 2003

A rat four vessel occlusion model was utilized to examine the effects of ischemia/reperfusion on cortical window superfusate levels of amino acids, glucose, and lactate. Superfusate aspartate, glutamate, phosphoethanolamine, taurine, and GABA were significantly elevated by cerebral ischemia, then declined during reperfusion. Other amino acids were affected to a lesser degree. Superfusate lactate rose slightly during the initial ischemic period, declined during continued cerebral ischemia and then was greatly elevated during reperfusion. Superfusate glucose levels declined to near zero levels during ischemia and then rebounded beyond basal levels during the reperfusion period. Inhibition of neuronal lactate uptake with α‐cyano‐4‐hydroxycinnamate dramatically elevated superfusate lactate levels, enhanced the ischemia/reperfusion evoked release of aspartate but reduced glutamine levels. Topical application of an alternative metabolic fuel, glutamine, had a dose dependent effect. Glutamine (1 mm) elevated basal superfusate glucose levels, diminished the decline in glucose during ischemia, and accelerated its recovery during reperfusion. Lactate levels were elevated during ischemia and reperfusion. These effects were not evident at 5 mmglutamine. At both concentrations, glutamine significantly elevated the superfusate levels of glutamate. Topical application of sodium pyruvate (20 mm) significantly attenuated the decline in superfusate glucose during ischemia and enhanced the levels of both glucose and lactate during reperfusion. However, it had little effect on the ischemia‐evoked accumulation of amino acids. Topical application of glucose (450 mg/dL) significantly elevated basal superfusate levels of lactate, which continued to be elevated during both ischemia and reperfusion. The ischemia‐evoked accumulations of aspartate, glutamate, taurine and GABA were all significantly depressed by glucose, while phosphoethanolamine levels were elevated. These results support the role of lactate in neuronal metabolism during ischemia/reperfusion. Both glucose and glutamine were also used as energy substrates. In contrast, sodium pyruvate does not appear to be as effectively utilized by the ischemic/reperfused rat brain since it did not reduce ischemia‐evoked amino acid efflux.
Integrins stimulate phosphorylation of neurofilament NF‐M subunit KSP repeats through activation of extracellular regulated‐kinases (Erk1/Erk2) in cultured motoneurons and transfected NIH 3T3 cells
Authors: Li Bing‐Sheng , Daniels Mathew P. , Pant Harish C.
Published: March 2003

Integrin‐mediated interactions of cells with components of the extracellular matrix (ECM) regulate cell survival, cell proliferation, cell differentiation and cell migration through activation of multiple intracellular signal transduction pathways. In this study, we have demonstrated that integrin–matrix interactions promote KSP tail‐domain phosphorylation of neurofilament medium molecular weight subunits (NF‐M) in cultured rat spinal cord motoneurons and NF‐M transfected NIH 3T3 cells. We found that laminin and fibronectin induce NF‐M tail‐domain phosphorylation in motoneurons and NIH 3T3 cells transfected with NF‐M, respectively. This phosphorylation was selectively inhibited by PD98059, a specific MEK1 inhibitor. This suggests that laminin and fibronectin‐induced MEK1 activation and the downstream targets Erk1 and Erk2 are involved in NF‐M KSP tail‐domain phosphorylation. This pathway appears to represent one of the mechanisms whereby integrin–extracellular matrix interactions are involved in phosphorylation of the NF‐M KSP tail domain.
Anti‐inflammatory effects of prostaglandin E2 in the central nervous system in response to brain injury and circulating lipopolysaccharide
Authors: Zhang Ji , Rivest Serge
Published: March 2003

Prostaglandin E2, a product of the cyclooxygenation of arachidonic acid released from membrane phospholipids, plays major roles in regulating brain injury and inflammation. Although prostaglandin E2 has frequently been considered as a possible inducer of brain damage and degeneration, it may exert beneficial effects in the CNS. Indeed, in spite of its classic role as a pro‐inflammatory molecule, several recent in vitro observations indicate that prostaglandin E2 can inhibit microglial activation. This study investigated the effect of central prostaglandin E2 injection on circulating lipopolysaccharide‐induced gene expression of different pro‐inflammatory molecules in both vascular and parenchymal elements of the brain. Localized, but strong, expression of tumor necrosis factor‐α and interleukin‐1β mRNA was found at the edge of the intracerebroventricular tract, which was largely prevented by the central prostaglandin E2 injection. Systemic lipopolysaccharide injection caused a profound transcriptional activation of cyclooxygenase‐2 and the inhibitory factor κBα (IκBα, index of NF‐κB activity) in the cerebral endothelium and tumor necrosis factor‐α in microglial cells across the brain parenchyma. Although exogenous prostaglandin E2 increased lipopolysaccharide‐induced NF‐κB activity and cyclooxygenase‐2 transcription in vascular‐associated elements, it significantly reduced microglial activation and tumor necrosis factor‐α expression in the brain parenchyma. These results indicate that prostaglandin E2 may play an important role in modulating the immune response occurring at the injured site and the pro‐inflammatory signaling events taking place in both vascular‐ and microglial‐associated elements of the CNS.
Cyclic ADP‐ribose as a potential second messenger for neuronal Ca2+ signaling
Authors: Higashida Haruhiro , Hashii Minako , Yokoyama Shigeru , Hoshi Naoto , Asai Kiyofumi , Kato Taiji
Published: March 2003

Cyclic ADP‐ribose (cADPR), a known endogenous modulator of ryanodine receptor Ca2+ releasing channels, is found in the nervous system. Injection of cADPR into neuronal cells primarily induces a transient elevation of intracellular Ca2+ concentration ([Ca2+]i), and/or secondarily potentiates [Ca2+]i increases that are the result of depolarization‐induced Ca2+ influx. Acetylcholine release from cholinergic neurons is facilitated by cADPR. cADPR modifies K+ currents or elicits Ca2+‐dependent inward currents. cADPR is synthesized by both membrane‐bound and cytosolic forms of ADP‐ribosyl cyclase in neuronal cells. cADPR hydrolase activity is weak in the membrane fraction, but high in the cytoplasm. Cytosolic ADP‐ribosyl cyclase activity is upregulated by nitric oxide/cyclic GMP‐dependent phosphorylation. Stimulation of muscarinic and β‐adrenergic receptors activates membrane‐bound ADP‐ribosyl cyclase via G proteins within membranes of neuronal tumor cells and cortical astrocytes. These findings strongly suggest that cADPR is a second messenger in Ca2+ signaling in the nervous system, although many intriguing issues remain to be addressed before this identity is confirmed.
Acute ethanol exposure induces [Ca2+]i transients, cell swelling and transformation of actin cytoskeleton in astroglial primary cultures
Authors: Allansson Louise , Khatibi Siamak , Olsson Torsten , Hansson Elisabeth
Published: March 2003

Acute exposure to 100 mm isotonic ethanol (EtOH) increased intracellular Ca2+ concentration ([Ca2+]i), induced cell swelling, and transformed actin cytoskeleton in astroglial primary cultures from rat cerebral cortex. Fluorometric recordings of fluo‐3AM‐ or fura‐2AM‐incubated astroglial cells revealed that EtOH induced [Ca2+]i transients in a small population of the cells. Cell swelling was estimated using a new method based on three‐dimensional fluorescence imaging in conjunction with image analysis and graphic visualization techniques. The method provides detailed results concerning the reformation of structural shape and specific volume alterations, as well as total proportions between the different states. Astroglial cell swelling was registered and quantified in 7 of 39 cells chosen from 12 different coverslips. EtOH also induced reversible conformational changes in filamentous actin, appearing as increases in ring formations and a more dispersed appearance of the filaments. Filamentous actin was stained with Alexa phalloidin after incubation with EtOH for varied periods. The results presented here suggest that EtOH affects astrocytes in a way that could be of physiological relevance.
Effect of overexpression of wild‐type and mutant Cu/Zn‐superoxide dismutases on oxidative damage and antioxidant defences: relevance to Down's syndrome and familial amyotrophic lateral sclerosis
Authors: Lee MoonHee , Hyun Dong‐Hoon , Jenner Peter , Halliwell Barry
Published: March 2003

Patients with Down's syndrome (DS) show elevated levels of copper, zinc‐containing superoxide dismutase (SOD1) and appear to have increased lipid peroxidation and oxidative damage to DNA as well as elevated glutathione peroxidase activity. Increasing SOD1 levels by gene transfection in NT‐2 and SK‐N‐MC cell lines also led to a rise in glutathione peroxidase activity, but this was nevertheless accompanied by decreased proliferation rates, increased lipid peroxidation and protein carbonyls, and a trend to a rise in 8‐hydroxyguanine and protein‐bound 3‐nitrotyrosine. Transfection of these cell lines with DNA encoding two mutant SOD1 enzymes (G37R and G85R) associated with familial amyotrophic lateral sclerosis (FALS), produced similar, but more severe changes, i.e. even lower growth rates, higher lipid peroxidation, 3‐nitrotyrosine and protein carbonyl levels, decreased GSH levels, raised GSSG levels and higher glutathione peroxidase activities. Since G85R has little SOD activity, these changes cannot be related to increased O2− scavenging. In no case was SOD2 (mitochondrial Mn‐SOD) level altered. Our cellular systems reproduce many of the biochemical changes observed in patients with DS or ALS, and in transgenic mice overexpressing mutant SOD1. They also show the potentially deleterious effects of SOD1 overexpression on cellular proliferation, which may be relevant to abnormal development in DS.
Stimulation of catecholamine synthesis in cultured bovine adrenal medullary cells by leptin
Authors: Utsunomiya Kensuke , Yanagihara Nobuyuki , Tachikawa Eiichi , Cheah Tat Beng , Kajiwara Koji , Toyohira Yumiko , Ueno Susumu , Izumi Futoshi
Published: March 2003

Recently, we characterized leptin receptors in bovine adrenal medullary cells (Yanagihara et al. 2000). Here we report the stimulatory effect of leptin on catecholamine synthesis in the cells. Incubating cells with leptin (10 nm) for 20 min increased the synthesis of 14C‐catecholamines from [14C]tyrosine, but not from l‐3,4‐dihydroxyphenyl [3–14C]alanine. The stimulation of catecholamine synthesis in the cells by leptin was associated with the phosphorylation and activation of tyrosine hydroxylase, the rate‐limiting enzyme of catecholamine biosynthesis. The incubation of cells with leptin resulted in a rapid activation of the mitogen‐activated protein kinases (MAPKs). An inhibitor of MAPK kinase, U0126, nullified the stimulatory effect of leptin on the synthesis of 14C‐catecholamines. Leptin potentiated the stimulatory effect of acetylcholine on 14C‐catecholamine synthesis, whereas leptin failed to enhance the phosphorylation and activation of tyrosine hydroxylase induced by acetylcholine. These findings suggest that leptin stimulates catecholamine synthesis via the activation of tyrosine hydroxylase by two different mechanisms, i.e., one is dependent on tyrosine hydroxylase phosphorylation mediated through the MAPK pathway and the second is independent of enzyme phosphorylation.
Nerve growth factor inhibits PC12 cell PDE 2 phosphodiesterase activity and increases PDE 2 binding to phosphoproteins
Authors: Bentley J. Kelley , Juilfs Dawn M. , Uhler Michael D.
Published: March 2003

Nerve growth factor (NGF) has been shown to increase cyclic AMP in PC12 cells and to potentiate the actions of other agents that raise cyclic AMP. In our studies, NGF causes over 50% loss of PDE 2 activity (cyclic GMP‐stimulated cyclic nucleotide phosphodiesterase) in PC12 cells within 24 h. After 72 h of NGF treatment, cyclic AMP hydrolysis in PC12 extracts is no longer cyclic GMP‐stimulated. NGF deprivation increases the phosphodiesterase activity of treated cells. NGF does not decrease either PDE 2 mRNA or immunoreactivity of PDE 2A2 protein. Incubation of whole cells with micromolar Na3VO4 mimics NGF treatment, reducing PDE 2 activity in PC12 cells by over 50% after 24 h, suggesting a phosphoprotein‐mediated regulation of PDE 2 activity. Protein kinase inhibitor effects were difficult to assess due to their direct interaction with the PDE in cell lysates. To study phosphorylation in PDE 2 regulation, PDE 2A2 was epitope‐tagged, and stable clonal PC12 cell transfectants were isolated (PC12B cells). When combined with metabolically labeled 32P‐phosphoproteins in vivo or in vitro, phosphoproteins of 108, 90, 64, 43, 33 and 19 kDa coprecipitated with epitope‐tagged PDE 2A2 in an NGF sensitive manner. A 23‐kDa phosphoprotein containing immunoreactive phosphoserine associated with the complex in an NGF independent manner. Phosphothreonine plus phosphotyrosine immunoreactivity at 23, 24, and 64 kDa as well as the phosphotyrosine immunoreactivity at 108, 90, 64, 43, 33, and 19 kDa required NGF or orthovanadate treatment. These proteins are hypothesized to be part of an NGF‐regulated complex controlling PDE 2A2 activity.
NF‐κB is involved in the survival of cerebellar granule neurons: association of Iκβ phosphorylation with cell survival
Authors: Koulich Elena , Nguyen Thuyen , Johnson Kyle , Giardina Charles A. , D'mello Santosh R.
Published: March 2003

The NF‐κB transcription factor consists of dimeric complexes belonging to the Rel family, which include p50, p52, p65 (RelA), RelB and c‐Rel. NF‐κB activity is tightly controlled by IκB proteins which bind to NF‐κB preventing its translocation to the nucleus. Activation of NF‐κB is most often mediated by IκB degradation, which permits NF‐κB to enter the nucleus. We investigated the role of NF‐κB in the survival of cerebellar granule neurons. We found that survival of these neurons in high potassium medium is blocked by three separate inhibitors of NF‐κB activity: SN‐50, N‐tosyl‐l‐phenylalanine chloromethyl ketone and pyrrolidinedithiocarbamate, indicating that NF‐κB is required for neuronal survival. Gel‐shift assays reveal three complexes that bind to the NF‐κB binding site in high potassium medium. Switching these cultures to low potassium medium, a stimulus that leads to apoptotic death, causes a reduction in the level of the largest complex, which contains p65. Overexpression of p65 by transfection inhibits low potassium‐induced apoptosis, whereas overexpression of IκBα promotes apoptosis even in high potassium medium. Surprisingly, however, neither the level of endogenous p65 nor that of IκBα and IκBβ is altered by low potassium treatment. Similarly, no changes are seen in the nuclear or cytoplasmic levels of p50, p52, RelB and c‐Rel. Phosphorylation of p65, which can lead to its activation, is unchanged. Phosphorylation of IκBβ is, however, reduced by low potassium treatment. Besides being necessary for high potassium‐mediated neuronal survival, NF‐κB is also involved in the survival‐promoting effects of IGF‐1 and cAMP as judged by the ability of SN‐50 to inhibit the actions of these survival factors and the ability of these factors to inhibit the low potassium‐induced alterations in the DNA‐binding activity of NF‐κB. Taken together, our results show that NF‐κB may represent a point of convergence in the signaling pathways activated by different survival factors and that uncommon mechanisms might be involved in NF‐κB‐mediated survival of cerebellar granule neurons.