bluuebunny/crossref_metadata_2025 huggingface dataset:
Long‐Term Depression in the Hippocampus In Vivo Is Associated with Protein Phosphatase‐Dependent Alterations in Extracellular Signal‐Regulated Kinase
Authors: Norman Eric D. , Thiels Edda , Barrionuevo German , Klann Eric
Published: March 2003

Abstract: There is growing evidence that activation of either protein kinases or protein phosphatases determines the type of plasticity observed after different patterns of hippocampal stimulation. Because activation of the extracellular signal‐regulated kinase (ERK) has been shown to be necessary for long‐term potentiation, we investigated the regulation of ERK in long‐term depression (LTD) in the adult hippocampus in vivo. We found that ERK immunoreactivity was decreased following the induction of LTD and that this decrease required NMDA receptor activation. The LTD‐associated decrease in ERK immunoreactivity could be simulated in vitro via incubation of either purified ERK2 or hippocampal homogenates with either protein phosphatase 1 or protein phosphatase 2A. The protein phosphatase‐dependent decrease in ERK immunoreactivity was inhibited by microcystin. Intrahippocampal administration of the protein phosphatase inhibitor okadaic acid blocked the LTD‐associated decrease in ERK2, but not ERK1, immunoreactivity. Collectively, these data demonstrate that protein phosphatases can decrease ERK immunoreactivity and that such a decrease occurs with ERK2 during LTD. These observations provide the first demonstration of a biochemical alteration of ERK in LTD.
The Effects of Serotonin on Glucocorticoid Receptor Binding in Rat Raphe Nuclei and Hippocampal Cells in Culture
Authors: Héry Micheline , Sémont Alexandra , Fache Marie‐Pierre , Faudon Maxime , Héry Francis
Published: March 2003

Abstract: The raphe‐hippocampal serotonin (5‐HT) system is involved in the regulation of the hypothalamuspituitary‐adrenal axis. The purpose of this study was to determine and compare the roles of 5‐HT in the regulation of glucocorticoid receptor (GR) binding in the raphe nuclei and in the hippocampus. The effects of 5‐HT, 5‐HT agonists, and the 5‐HT reuptake inhibitor citalopram on GR binding sites were studied in primary cultures of the fetal raphe nuclei and the hippocampus. Exposure of hippocampal cells to 5‐HT, (±)‐2,5‐dimethoxy‐4‐iodoamphetamine (DOI; a 5‐HT2 agonist), or citalopram resulted in an increase in number of GR binding sites. The effect of DOI was blocked by ketanserin (a 5‐HT2 antagonist). Specific and saturable GR binding was found in raphe cells. Exposure of raphe cells to 5‐HT, (±)‐8 hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT; a 5‐HT1A agonist), or citalopram induced a significant decrease in number of GR binding sites. The effect of 8‐OH‐DPAT was reversed by WAY 100135 [N‐tert‐butyl‐3‐[1‐[1‐(2‐methoxy)phenyl]piperazinyl]‐1‐phenylpropionamide; a 5‐HT1A antagonist]. These results show that the regulation of GRs during fetal life is structure‐dependent and involves different 5‐HT receptor subtypes. Moreover, the regulation of hippocampal GRs by citalopram suggests an action of antidepressants independent of their effects on monoamines.
Cyclic AMP‐Mediated Regulation of GABAA Receptor Subunit Expression in Mature Rat Cerebellar Granule Cells
Authors: Thompson C. L. , Razzini G. , Pollard S. , Stephenson F. A.
Published: March 2003

Abstract: Exposure of rat cerebellar granule cells cultured to
maturity in vitro to forskolin,
N6,2′‐O‐dibutyryladenosine
3′,5′‐cyclic monophosphate (Bt2cAMP), and
3‐isobutyl‐1‐methylxanthine (IBMX) down‐regulated GABAA receptor
α6 and β3 subunits but up‐regulated α1 and β2 subunits
with respect to vehicle‐treated controls. Dideoxyforskolin had no effect on
subunit expression. Protein kinase A inhibitors, H‐89 and
Rp‐adenosine 3′,5′‐cyclic monophosphothioate, prevented
these effects on α1 but not α6 subunit expression.
Flunitrazepam‐sensitive [3H]Ro 15‐4513 binding sites were increased
by 144 ± 20% following forskolin treatment. [3H]Ro 15‐4513
photoaffinity labelling showed that the GABAA receptor α1
subunit was the principal locus of the increased flunitrazepam‐sensitive
[3H]Ro 15‐4513 binding. Forskolin decreased
flunitrazepam‐insensitive [3H]Ro 15‐4513 binding sites by 25
± 8% and resulted in a 20% decrease in the irreversible incorporation
of radioactivity in the α6 subunit. Steady‐state levels of
GABAA receptor subunit mRNAs were determined by semiquantitative
RT‐PCR in forskolin‐treated cultures. Forskolin, Bt2cAMP, and IBMX
down‐regulated GABAA receptor α6 subunit mRNA expression;
α1 and β3 mRNA levels were unaffected, whereas β2 subunit mRNA
was up‐regulated. Dideoxy‐forskolin had no significant effect on α1,
α6, β2, and β3 mRNA levels. Thus, in mature
cerebellar granule cells, GABAA receptor expression can be regulated by intracellular cyclic AMP levels. This occurs at the level of gene transcription and/or translation by mechanisms that are only partially governed by protein kinase A.
Expression of 15 Glutamate Receptor Subunits and Various Splice Variants in Tissue Slices and Single Neurons of Brainstem Nuclei and Potential Functional Implications
Authors: Paarmann I. , Frermann D. , Keller B. U. , Hollmann M.
Published: March 2003

Abstract: Brainstem nuclei serve a diverse array of functions in
many of which ionotropic glutamate receptors are known to be involved.
However, little detailed information is available on the expression of
different glutamate receptor subunits in specific nuclei. We used RT‐PCR in
mice to analyze the glutamate receptor subunit composition of the
pre‐Bötzinger complex, the hypoglossal nucleus,
the nucleus of the solitary tract, and the inferior olive. Analyzing 15
receptor subunits and five variants, we found all four
α‐amino‐3‐hydroxy‐5‐methyl‐4‐propionic acid (AMPA) and six NMDA receptor
(NR) subunits as well as three of five kainate (KA) receptors (GluR5, GluR6,
and KA1) to be expressed in all nuclei. However, some distinct differences
were observed: The inferior olive preferentially expresses flop variants of
AMPA receptors, GluR7 is more abundant in the
pre‐Bötzinger complex than in the other nuclei,
and NR2C is most prominent in the nucleus of the solitary tract. In single
hypoglossal motoneurons and interneurons of the
pre‐Bötzinger complex investigation of GluR2
editing revealed strong expression of the GluR2‐R editing variant, suggesting
low Ca2+ permeability of AMPA receptors. Thus, Ca2+ ‐permeable AMPA receptors are unlikely to be the cause for the reported selective vulnerability of hypoglossal motoneurons during excitotoxic events.
Negligible Glucose‐6‐Phosphatase Activity in Cultured Astroglia
Authors: Gotoh Jun , Itoh Yoshiaki , Kuang Tang‐Yong , Cook Michelle , Law Mona J. , Sokoloff Louis
Published: March 2003

Abstract: 2‐Deoxy[14C]glucose‐6‐phosphate
(2‐[14C]DG‐6‐P) dephosphorylation and glucose‐6‐phosphatase
(G‐6‐Pase) activity were examined in cultured rat astrocytes under conditions
similar to those generally used in assays of glucose utilization. Astrocytes
were loaded with 2‐[14C]DG‐6‐P by preincubation for 15 min in
medium containing 2 mM glucose and 50 μM
2‐deoxy[14C]glucose (2‐[14C]DG). The medium was then
replaced with identical medium including 2 mM glucose but lacking
2‐[14C]DG, and incubation was resumed for 5 min to diminish
residual free 2‐[14C]DG levels in the cells by either efflux or
phosphorylation. The medium was again replaced with fresh
2‐[14C]DG‐free medium, and the incubation was continued for 5, 15,
or 30 min. Intracellular and extracellular 14C contents were
measured at each time point, and the distribution of 14C between
2‐[14C]DG and 2‐[14C]DG‐6‐P was characterized by paper
chromatography. The results showed little if any hydrolysis of
2‐[14C]DG‐6‐P or export of free 2‐[14C]DG from cells to
medium; there were slightly increasing losses of 2‐[14C]DG and
2‐[14C]DG‐6‐P into the medium with increasing incubation time, but
they were in the same proportions found in the cells, suggesting they were
derived from nonadherent or broken cells. Experiments carried out with medium
lacking glucose during the assay for 2‐deoxyglucose‐6‐phosphatase activity
yielded similar results. Evidence for G‐6‐Pase activity was also sought by
following the selective detritiation of glucose from the 2‐C position when
astrocytes were incubated with [2‐3H]glucose and
[U‐14C]glucose in the medium. No change in the
3H/14C ratio was found in incubations for as long as 15 min. These results indicate negligible G‐6‐Pase activity in cultured astrocytes.
Astrocytes Provide Cysteine to Neurons by Releasing Glutathione
Authors: Wang Xue Feng , Cynader Max S.
Published: March 2003

Abstract: Cysteine is the rate‐limiting precursor of glutathione synthesis. Evidence suggests that astrocytes can provide cysteine and/or glutathione to neurons. However, it is still unclear how cysteine is released and what the mechanisms of cysteine maintenance by astrocytes entail. In this report, we analyzed cysteine, glutathione, and related compounds in astrocyte conditioned medium using HPLC methods. In addition to cysteine and glutathione, cysteine‐glutathione disulfide was found in the conditioned medium. In cystine‐free conditioned medium, however, only glutathione was detected. These results suggest that glutathione is released by astrocytes directly and that cysteine is generated from the extracellular thiol/disulfide exchange reaction of cystine and glutathione: glutathione + cystine ↔ cysteine + cysteineglutathione disulfide. Conditioned medium from neuronenriched cultures was also assayed in the same way as astrocyte conditioned medium, and no cysteine or glutathione was detected. This shows that neurons cannot themselves provide thiols but instead rely on astrocytes. We analyzed cysteine and related compounds in rat CSF and in plasma of the carotid artery and internal jugular vein. Our results indicate that cystine is transported from blood to the CNS and that the thiol/disulfide exchange reaction occurs in the brain in vivo. Cysteine and glutathione are unstable and oxidized to their disulfide forms under aerobic conditions. Therefore, constant release of glutathione by astrocytes is essential to maintain stable levels of thiols in the CNS.
Microglial Apoptosis Induced by Chromogranin A Is Mediated by Mitochondrial Depolarisation and the Permeability Transition but Not by Cytochrome c Release
Authors: Kingham Paul J. , Pocock Jennifer M.
Published: March 2003

Abstract: Chromogranin A is up‐regulated in the senile plaques of
Alzheimer's brain and is a novel activator of microglia, transforming them to
a neurotoxic phenotype. Treatment of primary cultures of rat brain microglia
or the murine N9 microglial cell line with chromogranin A resulted in nitric
oxide production, which triggered microglial apoptosis. Exposure of microglia
to chromogranin A resulted in a fall in mitochondrial membrane potential.
Mitochondrial depolarisation and apoptosis were reduced significantly by
cyclosporin A, but not by the calcineurin inhibitor FK506. Cytochrome
c did not translocate from the mitochondria to the cytosol, but its
expression became significantly enhanced within the mitochondria. Inhibition
of caspase 1 attenuated chromogranin A‐induced microglial apoptosis, but did
not prevent mitochondrial depolarisation, indicating that apoptosis occurred
downstream of mitochondrial depolarisation. Conversely, staurosporine‐induced
microglial apoptosis led to mitochondrial cytochrome c release, but not caspase 1 activation. Our findings provide insight into the pathways controlling activation‐triggered microglial apoptosis and may point to routes for the modulation of microglial evoked neurotoxicity.
N‐Methyl Bases of Ethanolamine Prevent Apoptotic Cell Death Induced by Oxidative Stress in Cells of Oligodendroglia Origin
Authors: Brand Annette , Gil Shosh , Yavin Ephraim
Published: March 2003

Abstract: A major reason for brain tissue vulnerability to oxidative
damage is the high content of polyunsaturated fatty acids (PUFAs).
Oligodendroglia‐like OLN 93 cells lack PUFAs and are relatively insensitive to
oxidative stress. When grown in serum‐free defined medium in the presence of
0.1 mM docosahexaenoic acid (DHA; 22:6 n‐3) for 3 days, OLN 93 cells
release in the medium 2.6‐fold more thiobarbituric acid‐reactive substances
(TBARS) after a 30‐min exposure to 0.1 mM H2O2
and 50 μM Fe2+. Release of TBARS was substantially
decreased by ∼20 and 30% on coincubation with either 1 mMN‐monomethylethanolamine or N,N′‐dimethylethanolamine
(dEa), respectively. The protective effect of dEa was concentration‐ and
time‐dependent and was still visible after dEa removal, suggesting a
long‐lasting mechanism of protection. After 24 h following
H2O2‐induced stress, cell death monitored by cell
sorting showed 16% of the cells in the sub‐G1 area, indicative of apoptotic cell death. DHA‐supplemented cultures showed 35% cell death, whereas cosupplements with dEa reduced cell death to 12%, indicating cell rescue. Although the exact mechanism for this protection is not known, the nature of the polar head group and the degree of unsaturation may determine the ultimate resistance of nerve cells to oxidative stress.
Characterization in Cultured Cerebellar Granule Cells and in the Developing Rat Brain of mRNA Variants for the NMDA Receptor 2C Subunit
Authors: Rafiki Amina , Bernard Anne , Medina Igor , Gozlan Henri , Khrestchatisky Michel
Published: March 2003

Abstract:
N‐Methyl‐D‐aspartate (NMDA) receptors are heteromeric structures resulting from the association of at least two distantly related subunit types, NR1 and one of the four NR2 subunits (NR2A‐NR2D). When associated with NR1, the NR2 subunits impose specific properties to the reconstituted NMDA receptors. Although the NR1 mRNAs are expressed in the majority of central neurons, the NR2 subunits display distinct patterns of expression in the developing and adult rat brain. The NR2C subunit is barely expressed in the rat forebrain, whereas its expression increases substantially in the granule cells in the course of cerebellar development. We have identified novel NR2C splice variants in cultured cerebellar granule cells as well as in the developing cerebellum. When compared with the prototypic NR2C mRNA, these variants carry one (NR2Cb) or two (NR2Cd) insertions or a deletion (NR2Cc) and encode putative NR2C polypeptides that terminate between the third and fourth membrane segments or between the first and second membrane segments. RT‐PCR analysis and in situ hybridization show that expression of the splice variants is developmentally regulated, both in the cerebellum and in the hippocampus. Electrophysiological recordings and microfluorimetry emissions in transfected human embryonic kidney 293 cells indicate that the NR2Cb variant, when expressed in combination with NR1, does not contribute to the formation of functional receptor channels. The significance of these findings is discussed.
Efficient Gene Transfer and Expression of Biologically Active Glial Cell Line‐Derived Neurotrophic Factor in Rat Motoneurons Transduced with Lentiviral Vectors
Authors: Cisterni C. , Henderson C. E. , Aebischer P. , Pettmann B. , Déglon N.
Published: March 2003

Abstract:Several studies have shown the ability of human immunodeficiency virus type 1 (HIV1)‐based lentiviral vectors to infect nondividing brain and retinal neurons with high efficiency and long‐term expression of the transduced gene. We show that purified embryonic motoneurons can be efficiently (>95%) transduced in culture using an HIV1‐based lentiviral vector encoding LacZ. Expression of β‐galactosidase was observed for at least 9 days in these conditions. Furthermore, motoneurons transduced with a lentiviral vector expressing glial cell line‐derived neurotrophic factor survived in the absence of additional trophic support, showing that the overexpressed protein was biologically active. Our results demonstrate the potential of lentiviral vectors in studying the biological effects of proteins expressed in motoneurons and in the development of future gene therapy for motoneuron diseases.
Opposite Regulation of Calbindin and Calretinin Expression by Brain‐Derived Neurotrophic Factor in Cortical Neurons
Authors: Fiumelli Hubert , Kiraly Maria , Ambrus Andrea , Magistretti Pierre J. , Martin Jean‐Luc
Published: March 2003

Abstract: Regulation of calbindin and calretinin expression by brain‐derived neurotrophic factor (BDNF) was examined in primary cultures of cortical neurons using immunocytochemistry and northern blot analysis. Here we report that regulation of calretinin expression by BDNF is in marked contrast to that of calbindin. Indeed, chronic exposure of cultured cortical neurons for 5 days to increasing concentrations of BDNF (0.1‐10 ng/ml) resulted in a concentration‐dependent decrease in the number of calretinin‐positive neurons and a concentration‐dependent increase in the number of calbindin‐immunoreactive neurons. Consistent with the immunocytochemical analysis, BDNF reduced calretinin mRNA levels and up‐regulated calbindin mRNA expression, providing evidence that modifications in gene expression accounted for the changes in the number of calretinin‐ and calbindin‐containing neurons. Among other members of the neurotrophin family, neurotrophin‐4 (NT‐4), which also acts by activating tyrosine kinase TrkB receptors, exerted effects comparable to those of BDNF, whereas nerve growth factor (NGF) was ineffective. As for BDNF and NT‐4, incubation of cortical neurons with neurotrophin‐3 (NT‐3) also led to a decrease in calretinin expression. However, in contrast to BDNF and NT‐4, NT‐3 did not affect calbindin expression. Double‐labeling experiments evidenced that calretinin‐ and calbindin‐containing neurons belong to distinct neuronal subpopulations, suggesting that BDNF and NT‐4 exert opposite effects according to the neurochemical phenotype of the target cell.
Effect of Intracerebral Administration of NMDA and AMPA on Dopamine and Glutamate Release in the Ventral Pallidum and on Motor Behavior
Authors: Kretschmer Beate D. , Goiny Michel , Herrera‐Marschitz Mario
Published: March 2003

Abstract: The present study investigates the modulation of the ventral tegmental area (VTA)‐ventral pallidum (VP) dopaminergic system by glutamate agonists in rats. The glutamate receptor agonists N‐methyl‐D‐aspartate (NMDA) and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionic acid (AMPA) were infused via reversed microdialysis into the VTA, and dopamine (DA), glutamate, and aspartate levels in the VTA and ipsilateral VP were monitored together with motor behavior screened in an open field. NMDA (750 μM) infusion, as well as AMPA (50 μM) infusion, induced an increase of DA and glutamate levels in the VTA, followed by an increase of DA levels in the ipsilateral VP and by enhanced locomotor activity. The increase of DA in the VP was similar after administration of these two glutamate agonists, although motor activity was more pronounced and showed an earlier onset after NMDA infusion. Glutamate levels in the VP were not increased by the stimulation of DA release. It is concluded that DA is released from mesencephalic DA neurons projecting to the VP and that these neurons are controlled by glutamatergic systems, via NMDA and AMPA receptors. Thus, DA in the VP has to be considered as a substantial modulator. Dysregulation of the mesopallidal DA neurons, as well as their glutamatergic control, may play an additional or distinct role in disorders like schizophrenia and drug addiction.
Inhibition of Noxious Stimulus‐Induced Spinal Prostaglandin E2 Release by Flurbiprofen Enantiomers
Authors: Geisslinger Gerd , Muth‐Selbach Uta , Coste Ovidiu , Vetter Gregor , Schrödter Andreas , Schaible Hans‐Georg , Brune Kay , Tegeder Irmgard
Published: March 2003

Abstract: Peripheral noxious stimuli have been shown to induce prostaglandin (PG) E2 release at the site of inflammation and in the spinal cord. The antiinflammatory and antinociceptive effects of cyclooxygenase‐inhibiting drugs are thought to depend on the inhibition of PG synthesis. R‐Flurbiprofen, however, does not inhibit cyclooxygenase activity in vitro but still produces antinociceptive effects. To find out whether R‐flurbiprofen acts via inhibition of spinal PG release, concentrations of PGE2 and flurbiprofen in spinal cord tissue were assessed by microdialysis. The catheter was transversally implanted through the dorsal horns of the spinal cord at level L4. R‐ and S‐flurbiprofen (9 and 27 mg kg‐1, respectively) were administered intravenously 10‐15 min before subcutaneous injection of formalin into the dorsal surface of one hindpaw. Flurbiprofen was rapidly distributed into the spinal cord with maximal concentrations after 30‐45 min. Baseline PGE2 dialysate concentrations were 100.6 ± 6.4 pg ml‐1 (mean ± SEM). After formalin injection they rose about threefold with a maximum of 299.4 ± 68.4 pg ml‐1 at 7.5 min. After ∼ 1 h PGE2 levels returned to baseline. Both flurbiprofen enantiomers completely prevented the formalin‐induced increase of spinal PGE2 release and reduced PGE2 concentrations below basal levels. S‐ and R‐flurbiprofen at 9 mg kg‐1 produced a minimum of 15.8 ± 5.2 and 27.7 ± 14.9 pg ml‐1, respectively, and 27 mg kg‐1S‐ and R‐flurbiprofen resulted in 11.7 ± 1.7 and 9.3 ± 4.7 pg ml‐1, respectively. PGE2 levels remained at the minimum up to the end of the observation period at 5 h. When 27 mg kg‐1R‐flurbiprofen was injected intravenously without subsequent formalin challenge, baseline immunoreactive PGE2 concentrations were not affected. S‐Flurbiprofen (27 mg kg‐1), however, led to a moderate reduction (∼40%). The data suggest that antinociception produced by R‐flurbiprofen is mediated at least in part by inhibition of stimulated spinal PGE2 release and support the current view that increased spinal PGE2 release significantly contributes to nociceptive processing.
Increased Expression of Gαq/11 and of Phospholipase‐Cβ1/4 in Differentiated Human NT2‐N Neurons
Authors: Novak James E. , Agranoff Bernard W. , Fisher Stephen K.
Published: March 2003

Abstract: The CNS is enriched in phosphoinositide‐specific phospholipase C (PLC) and in the G proteins linked to its activation. Although the regional distributions of these signaling components within the brain have been determined, neither their cell type‐specific localizations (i.e., neuronal versus glial) nor the functional significance of their high expression has been definitively established. In this study, we have examined the expression of phosphoinositide signaling proteins in human NT2‐N cells, a well characterized model system for CNS neurons. Retinoic acid‐mediated differentiation of NT2 precursor cells to the neuronal phenotype resulted in five‐ to 15‐fold increases in the expression of PLC‐β1, PLC‐β4, and Gαq/11 (the prime G protein activator of these isozymes). In contrast, the expression of PLC‐β3 and PLC‐γ1 was markedly reduced following neuronal differentiation. Similar alterations in cell morphology and in the expression of PLC‐β1, PLC‐β3, and Gαq/11 expression were observed when NT2 cells were differentiated with berberine, a compound structurally unrelated to retinoic acid. NT2‐N neurons exhibited a significantly higher rate of phosphoinositide hydrolysis than NT2 precursor cells in response to direct activation of either G proteins or PLC. These results indicate that neuronal differentiation of NT2 cells is associated with dramatic changes in the expression of proteins of the phosphoinositide signaling system and that, accordingly, differentiated NT2‐N neurons possess an increased ability to hydrolyze inositol lipids.
Changes in NMDA Receptor Subunit Expression in Response to Contusive Spinal Cord Injury
Authors: Grossman Stacie D. , Wolfe Barry B. , Yasuda Robert P. , Wrathall Jean R.
Published: March 2003

Abstract : Differential assembly of N‐methyl‐D‐aspartate (NMDA) receptor subunits determines their functional characteristics. Using in situ hybridization, we found a selective increase of the subunits NR1 and NR2A mRNA at 24 h in ventral motor neurons (VMN) caudal to a standardized spinal cord contusion injury (SCI). Other neuronal cell populations and VMN rostral to the injury site appeared unaffected. Significant up‐regulation of NR2A mRNA also was seen 1 month after SCI in thoracic and lumbar VMN. The selective effects on VMN caudal to the injury site suggest that the loss of descending innervation leads to increased NMDA receptor subunit expression in these cells after SCI, which may alter their responses to glutamate. In contrast, protein levels determined by western blot analysis show decreased levels of NR2A 1 month after SCI in whole thoracic segments of spinal cord that included the injury sites. No effects of injury were seen on subunit levels in cervical or lumbar segments. Taken together with our previous study showing α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate receptor subunit down‐regulation after injury, our data suggest that glutamate receptor composition is significantly altered after SCI. These changes need to be taken into account to properly understand the function of, and potential pharmacotherapy for, the chronically injured spinal cord.
Metallothionein‐III Prevents Glutamate and Nitric Oxide Neurotoxicity in Primary Cultures of Cerebellar Neurons
Authors: Montoliu Carmina , Monfort Pilar , Carrasco Javier , Palacios Óscar , Capdevila Mercé , Hidalgo Juan , Felipo Vicente
Published: March 2003

Abstract : Metallothionein (MT)‐III, a member of the MT family of

metal‐binding proteins, is mainly expressed in the CNS and is abundant in

glutamatergic neurons. Results in genetically altered mice indicate that

MT‐III may play neuroprotective roles in the brain, but the mechanisms through

which this protein functions have not been elucidated. The aim of this work

was to assess whether MT‐III is able to prevent glutamate neurotoxicity and to

identify the step of the neurotoxic process interfered with by MT‐III.

Glutamate neurotoxicity in cerebellar neurons in culture is mediated by

excessive activation of glutamate receptors, increased intracellular calcium,

and increased nitric oxide. It is shown that MT‐III prevented glutamate‐ and

nitric oxide‐induced neurotoxicity in a dose‐dependent manner, with nearly

complete protection at 0.3‐1 μg/ml. MT‐III did not prevent the

glutamate‐induced rise of intracellular calcium level but reduced

significantly the nitric oxide‐induced formation of cyclic GMP. Circular

dichroism analysis revealed that nitric oxide triggers the release of the

metals coordinated to the cysteine residues of MT‐III, indicative of the

SCys‐nitrosylation of the protein. Therefore, the present results indicate that MT‐III can quench pathological levels of nitric oxide, thus preventing glutamate and nitric oxide neurotoxicity.
Tyrosine Phosphorylation of PNS Myelin P0 Occurs in the Cytoplasmic Domain and Is Maximal During Early Development
Authors: Iyer Srinivas , Bianchi Roberto , Eichberg Joseph
Published: March 2003

Abstract P0, the major protein of PNS myelin, is

considered to play a critical role in the compaction and stabilization of

myelin lamellae. The protein undergoes extensive posttranslational

modifications, including phosphorylation at multiple serine moieties in the

cytoplasmic region. Recently, we demonstrated that P0 is

phosphorylated on one or more tyrosine residues in rat nerve homogenates after

incubation. In this study, we show that P0 phosphorylated on

tyrosine is also present in the intact animal. The proportion of P0

molecules phosphorylated on tyrosine is highest during the first postnatal

week, a period that coincides with the most rapid period of myelin deposition

in the PNS. A peptide that constitutes the cytoplasmic domain was isolated

from purified P0 and shown by immunochemical and chemical means to

be phosphorylated on the tyrosine corresponding to Y191 in the

intact protein. No evidence was obtained supporting the possibility that

P0 is phosphorylated on other tyrosine residues. The sequence of

amino acids surrounding Y191 resemble known substrate phosphorylation sites for some nonreceptor cytoplasmic tyrosine kinases, as well as tyrosine‐based recognition signals associated with clathrin vesicle‐mediated cndocytosis.
Effects of Oxidative Stress on Phospholipid Signaling in Rat Cultured Astrocytes and Brain Slices
Authors: Servitja Joan‐Marc , Masgrau Roser , Pardo Raúl , Sarri Elisabet , Picatoste Fernando
Published: March 2003

Although reactive oxygen species (ROS) are conventionally viewed as toxic by‐products of cellular metabolism, a growing body of evidence suggests that they may act as signaling molecules. We have studied the effects of hydrogen peroxide (H2O2)‐induced oxidative stress on phospholipid

signaling in cultured rat cortical astrocytes. H2O2

stimulated the formation of phosphatidic acid and the accumulation of

phosphatidylbutanol, a product of the phospholipase D (PLD)‐catalyzed

transphosphatidylation reaction. The effect of exogenous

H2O2 on the PLD response was mimicked by

menadione‐induced production of endogenous H2O2.

Oxidative stress also elicited inositol phosphate accumulation resulting from

phosphoinositide phospholipase C (PLC) activation. The PLD response to

H2O2 was totally suppressed by chelation of both

extracellular and cytosolic Ca2+ with EGTA and BAPTA/AM,

respectively. Furthermore, H2O2‐induced PLD stimulation

was completely abolished by the protein kinase C (PKC) inhibitors

bisindolylmaleimide and chelerythrine and by PKC down‐regulation. Activation

of PLD by H2O2 was also inhibited by the

protein‐tyrosine kinase inhibitor genistein. Finally,

H2O2 also stimulated both PLC and PLD in rat brain cortical slices. These results show for the first time that oxidative stress elicits phospholipid breakdown by both PLC and PLD in rat cultured astrocytes and brain slices.
Astroglial Trophic Support and Neuronal Cell Death
Authors: Ohgoh Makoto , Shimizu Hiroki , Ogura Hiroo , Nishizawa Yukio
Published: March 2003

Abstract: Previous in vivo and in vitro analyses have shown that both necrosis and apoptosis are involved in neuronal cell death induced by energy impairment caused by mitochondrial dysfunction. However, little is known about the key factors that determine whether the cells undergo necrosis or apoptosis. In the present study, we analyzed neuronal cell death induced by 3‐nitropropionic acid (3‐NP), an irreversible inhibitor of mitochondrial complex II, in a primary culture system of rat cortical neurons. The neurons were maintained for a week in coculture with astroglial cells, and then they were treated with 3‐NP in the presence or absence of astroglial cells. As judged from morphological (Hoechst 33258 staining) and biochemical (DNA fragmentation and caspase activation) analyses, the cortical neurons appeared to die through an apoptotic process after 3‐NP treatment in the presence of astroglial cells. However, caspase inhibitors did not suppress the 3‐NP‐induced cell death, suggesting the involvement of a caspase‐independent pathway of 3‐NP‐induced neuronal cell death in the presence of astroglial cells. On the other hand, 3‐NP induced necrotic cell death within 1 day in the absence of astroglial cells, following a rapid decrease in intracellular ATP level. These changes were attenuated by the presence of astroglial cells or the addition of astroglial conditioned medium. These results suggest that astroglial trophic support influences the alteration of the intracellular energy state in 3‐NP‐treated neurons and consequently determines the type of neuronal cell death, apoptosis or necrosis.
Cisplatin‐Induced Apoptotic Cell Death in Mouse Hybrid Neurons Is Blocked by Antioxidants Through Suppression of Cisplatin‐Mediated Accumulation of p53 but Not of Fas/Fas Ligand
Authors: Park Sun Ah , Choi Kyeong Sook , Bang Jung Hee , Huh Kyoon , Kim Seung U.
Published: March 2003

Abstract: Peripheral neuropathy following cisplatin treatment is a

major limiting factor in cisplatin chemotherapy of cancer patients. We

investigated the pathomechanism underlying cisplatin neuropathy using a mouse

dorsal root ganglion neuron‐neuroblastoma hybrid cell line (N18D3) developed

in our laboratory. DNA fragmentation, a characteristic feature of apoptosis,

was induced in hybrid neurons following treatment with cisplatin. Accumulation

of p53, Fas, and Fas ligand (Fas‐L) was also demonstrated in these neurons.

Preincubation with N‐acetylcysteine (NAC), a precursor of glutathione, blocked cisplatin‐induced apoptosis completely, whereas Trolox, a vitamin E analogue, blocked it partially. Cisplatin‐induced p53 accumulation was suppressed by NAC treatment, whereas p53 accumulation was retarded by Trolox treatment. In contrast, neither NAC nor Trolox showed any inhibitory effect on cisplatin‐induced Fas/Fas‐L accumulation. These results suggest that the neuroprotective effects of antioxidants against cisplatin‐induced neurotoxicity in hybrid neurons are mediated mainly through the inhibition of p53 accumulation but not of Fas/Fas‐L accumulation by these antioxidants.
Diazepam Promotes ATP Recovery and Prevents Cytochrome c Release in Hippocampal Slices After In Vitro Ischemia
Authors: Galeffi Francesca , Sinnar Shamim , Schwartz‐Bloom Rochelle D.
Published: March 2003

Abstract: Benzodiazepines protect hippocampal neurons when

administered within the first few hours after transient cerebral ischemia.

Here, we examined the ability of diazepam to prevent early signals of cell

injury (before cell death) after in vitro ischemia. Ischemia in vitro or in

vivo causes a rapid depletion of ATP and the generation of cell death signals,

such as the release of cytochrome c from mitochondria. Hippocampal

slices from adult rats were subjected to 7 min of oxygen‐glucose deprivation

(OGD) and assessed histologically 3 h after reoxygenation. At this time, area

CA1 neurons appeared viable, although slight abnormalities in structure were

evident. Immediately following OGD, ATP levels in hippocampus were decreased

by 70%, and they recovered partially over the next 3 h of reoxygenation. When

diazepam was included in the reoxygenation buffer, ATP levels recovered

completely by 3 h after OGD. The effects of diazepam were blocked by

picrotoxin, indicating that the protection was mediated by an influx of

Cl‐ through the GABAA receptor. It is interesting that

the benzodiazepine antagonist flumazenil did not prevent the action of

diazepam, as has been shown in other studies using the hippocampus. Two hours

after OGD, the partial recovery of ATP levels occurred simultaneously with an

increase of cytochrome c (∼400%) in the cytosol. When diazepam

was included in the reoxygenation buffer, it completely prevented the increase

in cytosolic cytochrome c. Thus, complete recovery of ATP and

prevention of cytochrome c release from mitochondria can be achieved when diazepam is given after the loss of ATP induced by OGD.
The FGFR1 Inhibitor PD 173074 Selectively and Potently Antagonizes FGF‐2 Neurotrophic and Neurotropic Effects
Authors: Skaper Stephen D. , Kee Wai Jing , Facci Laura , Macdonald Gregor , Doherty Patrick , Walsh Frank S.
Published: March 2003

Abstract: Basic fibroblast growth factor (FGF‐2) promotes survival
and/or neurite outgrowth from a variety of neurons in cell culture and
regenerative processesin vivo. FGFs exert their effects by activating cell
surface receptor tyrosine kinases. FGF receptor (FGFR) inhibitors have not
been characterized on neuronal cell behaviors to date. In the present study,
we show that the FGFR1 inhibitor PD 173074 potently and selectively
antagonized the neurotrophic and neurotropic actions of FGF‐2. Nanomolar
concentrations of PD 173074 prevented FGF‐2, but not insulin‐like growth
factor‐1, support of cerebellar granule neuron survival under conditions of
serum/K+ deprivation; another FGF‐2 inhibitor, SU 5402, was
effective only at a 1,000‐fold greater concentration. Neither PD 173074 nor SU
5402, at 100 times their IC50 values, interfered with the survival
of dorsal root ganglion neurons promoted by nerve growth factor, ciliary
neurotrophic factor, or glial cell line‐derived neurotrophic factor. PD 173074
and SU 5402 displayed 1,000‐fold differential IC50 values for inhibition of FGF‐2‐stimulated neurite outgrowth in PC12 cells and in granule neurons, and FGF‐2‐induced mitogen‐activated protein kinase (p44/42) phosphorylation. The two inhibitors failed to disturb downstream signalling stimuli of FGF‐2. PD 173074 represents a valuable tool for dissecting the role of FGF‐2 in normal and pathological nervous system function without compromising the actions of other neurotrophic factors.
Glycosphingolipid Composition of a New Immortalized Human Cerebromicrovascular Endothelial Cell Line
Authors: Duvar Sevim , Suzuki Minoru , Muruganandam Arumugam , Yu Robert K.
Published: March 2003

Abstract: Previous studies have demonstrated the involvement of glycosphingolipid (GSL) antigens in the pathogenesis of immune‐mediated neurological disorders such as peripheral neuropathies and multiple sclerosis. To study the role of the blood‐brain barrier (BBB) in these disorders, we used a new human cerebromicrovascular endothelial cell (HCEC) line that has been immortalized through transfection with the plasmid pSV3‐neo encoding for the SV40 large T‐antigen and the neomycin gene. The immortalized HCEC (SV‐HCEC) exhibited accelerated proliferation rates but maintained phenotypic properties of early‐passage control cells. Therefore, this human cell line may serve as a useful in vitro model for studying the properties of the human BBB. We first investigated the GSL composition of cultured SV‐HCECs. The major gangliosides were GM3 (62% of total gangliosides), GM2 (18%), GM1 (3%), and GD1a (15%). The major neutral GSLs were glucosylceramide (15% of the total neutral glycolipids), lactosylceramide (36%), globotriaosylceramide (3%), and globoside (43%). Trace amounts of paragloboside, lactosaminyl paragloboside, and sulfoglucuronyl paragloboside could also be detected by TLC‐immunostaining. These results provide the basis for further investigations of the expression of these cell surface antigens in cultured SV‐HCECs on activation with inflammatory cytokines such as interleukin‐1β, tumor necrosis factor‐α, and interferon‐γ, which have been implicated as playing an important role in the pathogenesis of many nervous system disorders.
Tissue Plasminogen Activator Requires Plasminogen to Modulate Amyloid‐β Neurotoxicity and Deposition
Authors: Tucker H. Michael , Kihiko‐Ehmann Muthoni , Wright Sarah , Rydel Russell E. , Estus Steven
Published: March 2003

Abstract: Tissue plasminogen (plgn) activator (tPA) modulates neuronal death in models of stroke, excitotoxicity, and oxidative stress. Amyloid‐β (Aβ) appears central to Alzheimer's disease and is neurotoxic to neurons in vitro. Here, we evaluate tPA effects on Aβ toxicity. We report that tPA alone had no effect on Aβ toxicity. However, in combination with plgn, tPA reduced Aβ toxicity in a robust fashion. Moreover, the combined tPA and plgn treatment markedly inhibited Aβ accumulation. The addition of phenylmethylsulfonyl fluoride, a serine protease inhibitor, to a sample of tPA, plgn, and Aβ resulted in a marked reduction of Aβ degradation. We interpret the actions of tPA and plgn within the context of the ability of plasmin to degrade Aβ.
Functional Role of Tryptophan Residues in the Fourth Transmembrane Domainof the CB2 Cannabinoid Receptor
Authors: Rhee Man‐Hee , Nevo Igal , Bayewitch Michael L. , Zagoory Orna , Vogel Zvi
Published: March 2003

Abstract: Several tryptophan (Trp) residues are conserved in Gprotein‐coupled receptors (GPCRs). Relatively little is known about thecontribution of these residues and especially of those in the fourthtransmembrane domain in the function of the CB2 cannabinoidreceptor. Replacing W158 (very highly conserved in GPCRs) and W172 (conservedin CB1 and CB2 cannabinoid receptors but not in manyother GPCRs) of the human CB2 receptor with A or L or with F or Yproduced different results. We found that the conservative change of W172 to For Y retained cannabinoid binding and downstream signaling (inhibition ofadenylyl cyclase), whereas removal of the aromatic side chain by mutating W172to A or L eliminated agonist binding. W158 was even more sensitive to beingmutated. We found that the conservative W158F mutation retained wild‐typebinding and signaling activities. However, W158Y and W158A mutants completelylost ligand binding capacity. Thus, the Trp side chains at positions 158 and172 seem to have a critical, but different, role in cannabinoid binding to thehuman CB2 receptor.
“Inflammatory” Cytokines
Authors: Vitkovic Ljubisa , Bockaert Joël , Jacque Claude
Published: March 2003

Abstract: If cytokines are constitutively expressed by and act on neurons in normal adult brain, then we may have to modify our current view that they are predominantly inflammatory mediators. We critically reviewed the literature to determine whether we could find experimental basis for such a modification. We focused on two “proinflammatory” cytokines, interleukin (IL)‐1 and tumor necrosis factor‐α (TNFα) because they have been most thoroughly investigated in shaping our current thinking. Evidence, although equivocal, indicates that the genes coding for these cytokines and their accessory proteins are expressed by neurons, in addition to glial cells, in normal brain. Their expression is region‐ and cell type‐specific. Furthermore, bioactive cytokines have been extracted from various regions of normal brain. The cytokines’ receptors selectively are present on all neural cell types, rendering them responsive to cytokine signaling. Blocking their action modifies multiple neural “house‐keeping” functions. For example, blocking IL‐1 or TNFα by several independent means alters regulation of sleep. This indicates that these cytokines likely modulate in the brain behavior of a normal organism. In addition, these cytokines are likely involved in synaptic plasticity, neural transmission, and Ca2+ signaling. Thus, the evidence strongly suggests that these cytokines perform neural functions in normal brain. We therefore propose that they should be thought of as neuromodulators in addition to inflammatory mediators.
Down‐Regulation of GD3 Ganglioside and Its O‐Acetylated Derivative by Stable Transfection with Antisense V
Authors: Birklé Stéphane , Gao Luoyi , Zeng Guichao , Yu Robert K.
Published: March 2003

Abstract: The expression of gangliosides in hamster melanoma cells is closely related to cellular growth and degree of differentiation, with slow‐growing, highly differentiated melanotic melanoma cells expressing GM3 and fast‐growing, undifferentiated amelanotic Ab melanoma cells having a preponderance of GD3 and O‐acetyl‐GD3. We recently showed that down‐regulation of O‐acetyl‐GD3 expression in hamster melanoma cells by introducing the influenza C virus O‐acetylesterase cDNA into the cells resulted in induction of dendricity, with a concomitant increased expression of GD3. To examine the effect of the increased GD3 expression in the plasma membrane on the dendricity of the AbC‐1 cells, we first established the cDNA coding for hamster GD3‐synthase. We then targeted the sialyltransferase gene expression by the antisense knockdown experiment, and the results showed that inhibition of the expression of gangliosides GD3 and O‐acetyl‐GD3 induced dendricity in the hamster melanoma AbC‐1 cell line. These GD3‐ and O‐acetyl‐GD3‐depleted cells also demonstrated a decreased rate of cell growth, but their melanogenic potential was not affected. These results rule out the possibility that GD3 may serve as an active molecule for dendrite outgrowth in this cell line and suggest that the enhanced expression of O‐acetyl‐GD3 ganglioside may stimulate cellular growth and suppress certain differentiated phenotypes such as dendrite formation, but not melanogenesis, in our system.
μ‐Opioid Agonist Inhibition of κ‐Opioid Receptor‐Stimulated Extracellular Signal‐Regulated Kinase Phosphorylation Is Dynamin‐Dependent in C6 Glioma Cells
Authors: Bohn Laura M. , Belcheva Mariana M. , Coscia Carmine J.
Published: March 2003

Abstract: In previous studies we found that μ‐opioids, acting via μ‐opioid receptors, inhibit endothelin‐stimulated C6 glioma cell growth. In the preceding article we show that the κ‐selective opioid agonist U69,593 acts as a mitogen with a potency similar to that of endothelin in the same astrocytic model system. Here we report that C6 cell treatment with μ‐opioid agonists for 1 h results in the inhibition of κ‐opioid mitogenic signaling. The μ‐selective agonist endomorphin‐1 attenuates κ‐opioid‐stimulated DNA synthesis, phosphoinositide turnover, and extracellular signal‐regulated kinase phosphorylation. To investigate the role of receptor endocytosis in signaling, we have examined the effects of dynamin‐1 and its GTPase‐defective, dominant suppressor mutant (K44A) on opioid modulation of extracellular signal‐regulated kinase phosphorylation in C6 cells. Overexpression of dynamin K44A in C6 cells does not affect κ‐opioid phosphorylation of extracellular signal‐regulated kinase. However, it does block the inhibitory action on κ‐opioid signaling mediated by the κ‐opioid receptor. Our results are consistent with a growing body of evidence of the opposing actions of μ‐ and κ‐opioids and provide new insight into the role of opioid receptor trafficking in signaling.
The Butyrylcholinesterase K‐Variant Shows Similar Cellular Protein Turnover and Quaternary Interaction to the Wild‐Type Enzyme
Authors: Altamirano Cibby Varkey , Bartels Cynthia F. , Lockridge Oksana
Published: March 2003

Abstract: A recent study has linked the butyrylcholinesterase (BChE) K‐variant and the apolipoprotein ε4 isoform to late‐onset Alzheimer’s disease. These findings have been controversial and have led us to examine the differences between wild‐type and K‐variant BChE in enzyme activity, protein stability, and quaternary structure. J‐variant BChE (E497V/A539T) was also studied because it is associated with the K‐variant mutation. The K‐variant mutation (A539T) is located in the C‐terminal tetramerization domain. Wild‐type, K‐variant, and J‐variant BChE were expressed in Chinese hamster ovary cells and purified. The purified enzymes had similar binding affinity (Km) values and catalytic rates for butyrylthiocholine and benzolycholine. In pulse‐chase studies the K‐variant, J‐variant, and wild‐type BChE were degraded rapidly within the cell, with a half‐time of ∼ 1.5 h. Less than 5% of the intracellular BChE was exported. The C‐terminal peptide containing the K‐variant mutation interacted with itself as strongly as did the wild‐type peptide in the yeast two‐hybrid system. Both K‐variant and wild‐type BChE assembled into tetramers in the presence of poly‐L‐proline or the proline‐rich attachment domain of the collagen tail. The native K‐variant BChE in serum showed the same proportion of tetramers as the native serum wild‐type BChE. We conclude that the K‐variant BChE is similar to wild‐type BChE in enzyme activity, protein turnover, and tetramer formation.