bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunologically Distinct Isoforms of Ecto‐5′‐Nucleotidase in Nerve Terminals of Different Areas of the Rat Hippocampus
Authors: Cunha Rodrigo A. , Brendel Peter , Zimmermann Herbert , Ribeiro J. A.
Published: March 2003

Abstract: Ecto‐5′‐nucleotidase is regarded as being the key enzyme in the formation of the neuromodulator adenosine from released ATP. However, the association of ecto‐5′‐nucleotidase with nerve terminals is not consensual. Only enzyme histochemical and biochemical studies, but not immunocytochemical studies, agree on a general synaptic location of the enzyme. To clarify this issue further we tested the effect of an antibody against ecto‐5′‐nucleotidase, previously used in immunocyto‐chemical studies, on the activity of ecto‐5′‐nucleotidase in fractions of nerve terminals isolated from different areas of rat hippocampus. The specific activity of extracellular AMP catabolism was higher in synaptosomes from the CA3 area (0.81 ± 0.06 nmol/min/mg of protein) than from synaptosomes from the CA1 area or the dentate gyrus or from the whole hippocampus (0.49‐0.68 nmol/min/mg of protein). The catabolism of AMP (10 μM) was equally inhibited (85‐92%) in synaptosomes from whole hippocampus, CA1, CA3, or dentate gyrus by α,β‐methylene‐ADP (100 μM) and equally unaffected by p‐nitrophenyl phosphate (0.5 mM) or rabbit IgGs (100 μg/ml). However, the antiserum against ecto‐5′‐nucleotidase (100 μg/ml) inhibited extracellular AMP catabolism by 44% in CA3 synaptosomes but had little or no effect in synaptosomes from CA1, dentate gyrus, or whole hippocampus. A similar difference in the inhibitory potential of the antibody was observed between fractions of isolated 5′‐nucleotidase binding to concanavalin A‐Sepharose (70%) and fractions not retained by the lectin column (18%). Taken together, these results suggest that immunological isoforms of ecto‐5′‐nucleotidase exist in the rat hippocampal nerve terminals, with predominance in the CA3 area.
Multiple Calcium Pathways Induce the Expression of SNAP‐25 Protein in Chromaffin Cells
Authors: García‐Palomero Esther , Montiel Carmen , Herrero Carlos J. , García Antonio G. , Alvarez Rocio M. , Arnalich Francisco M. , Renart Jaime , Lara Hernan , Cárdenas Ana M.
Published: March 2003

Abstract: Incubation of bovine adrenal chromaffin cells in high
K+ (38 mM) during 24‐48 h enhanced 2.5 to five times the
expression of SNAP‐25 protein and mRNA, respectively. This increase was
reduced 86% by furnidipine (an L‐type Ca2+ channel blocker) but was
unaffected by either ω‐conotoxin GVIA (an N‐type Ca2+ channel
blocker) or ω‐agatoxin IVA (a P/Q‐type Ca2+ channel blocker).
Combined blockade of N and P/Q channels with ω‐conotoxin MVIIC did,
however, block by 76% the protein expression. The inhibitory effects of
furnidipine were partially reversed when the external Ca2+
concentration was raised from 1.6 to 5 mM. These findings, together
with the fact that nicotinic receptor activation or Ca2+ release
from internal stores also enhanced SNAP‐25 protein expression, suggest that an
increment of cytosolic Ca2+ concentration
([Ca2+]i), rather than its source or Ca2+
entry pathway, is the critical signal to induce the protein expression. The
greater coupling between L‐type Ca2+ channels and protein
expression might be due to two facts: (a) L channels contributed 50% to the
global [Ca2+]i rise induced by 38 mM
K+ in indo‐1‐loaded chromaffin cells and (b) L channels undergo less inactivation than N or P/Q channels on sustained stimulation of these cells.
Zinc and Copper Modulate Differentially the P2X4 Receptor
Authors: Acuña‐Castillo Claudio , Morales Bernardo , Huidobro‐Toro J. Pablo
Published: March 2003

Abstract: The rat ATP P2X4 receptor was expressed in
Xenopus laevis oocytes to assess the effect of zinc and copper as
possible regulators of purinergic mechanisms. ATP applied for 20 s evoked an
inward cationic current with a median effective concentration
(EC50) of 21.4 ± 2.8 μM and a Hill coefficient
(nH) of 1.5 ± 0.1. Coapplication of ATP plus 10
μM zinc displaced leftward, in a parallel fashion, the ATP
concentration‐response curve, reducing the EC50 to 8.4 ± 1.8
μM (p < 0.01) without altering the receptor
nH. The zinc potentiation was fast in onset, easily
reversible, and voltage‐independent and did not require metal preexposure. The
zinc EC50 was 2‐5 μM, with a bell‐shaped curve. At
concentrations of 100‐300 μM, zinc produced less potentiation, and
at 1 mM, it inhibited 50% the ATP current. The effect of zinc was
mimicked by cadmium. In contrast, copper inhibited the ATP‐evoked currents in
a time‐ and concentration‐dependent fashion, reducing the maximal current
(Imax) without altering the EC50. The
copper‐induced inhibition was slow in onset, slowly reversible, and
voltage‐independent. Whereas coapplication of 300 μM copper plus
ATP reduced Imax to 36.2 ± 5%, the coapplication
of, or 60‐s preexposure by, 10 μM copper reduced
Imax to 79 ± 9.2% (p < 0.05) and 39.6
± 8.7% (p < 0.01), respectively. The inhibition was
noncompetitive in nature and mimicked by mercury. Cobalt, barium, and
manganese did not modify significantly the ATP‐evoked current, demonstrating
metal specificity. The simultaneous 1‐min preapplication of both metals
revealed that the 10 μM zinc‐induced potentiation was obliterated
by 10 μM copper, whereas 30 μM copper not only reduced
the potentiation, but inhibited the ATP response. Following coapplication of
both metals for 20 s with ATP, at least 100 μM copper was required
to counteract the 10 μM zinc‐induced potentiation. The
simultaneous preincubation with both metals provided evidence for a
noncompetitive interaction. We hypothesize the existence of metal binding
site(s), which are most likely localized in the extracellular domain of the
P2X4 receptor structure. These sites are selective and accessible
to extracellular metal applications and bind micromolar concentrations of
metals. The present results are compatible with the working hypothesis that
trace metals, such as copper and zinc, are physiological modulators of the
P2X4 receptor. The modulation of brain purinergic transmission by physiologically and toxicologically relevant trace metal cations is highlighted.
Phosphorylation Sites on Tau Identified by Nanoelectrospray Mass Spectrometry
Authors: Reynolds C. Hugh , Betts Joanna C. , Blackstock Walter P. , Nebreda Angel R. , Anderton Brian H.
Published: March 2003

Abstract: The stress‐activated kinases c‐Jun N‐terminal kinase (JNK)
and p38 are members of the mitogen‐activated protein (MAP) kinase family and
take part in signalling cascades initiated by various forms of stress. Their
targets include the microtubule‐associated protein tau, which becomes
hyperphosphorylated in Alzheimer's disease. It is necessary, as a forerunner
for in vivo studies, to identify the protein kinases and phosphatases that are
responsible for phosphate turnover at individual sites. Using nanoelectrospray
mass spectrometry, we have undertaken an extensive comparison of
phosphorylation in vitro by several candidate tau kinases, namely, JNK, p38,
ERK2, and glycogen synthase kinase 3β (GSK3β). Between 10 and 15
sites were identified for each kinase. The three MAP kinases phosphorylated
Ser202 and Thr205 but not detectably Ser199,
whereas conversely GSK3β phosphorylated Ser199 but not
detectably Ser202 or Thr205. Phosphorylated
Ser404 was found with all of these kinases except JNK. The MAP
kinases may not be strictly proline specific: p38 phosphorylated the
nonproline sites Ser185, Thr245, Ser305, and
Ser356, whereas ERK2 was the most strict. All of the sites detected
except Thr245 and Ser305 are known or suspected phosphorylation sites in paired helical filament‐tau extracted from Alzheimer brains. Thus, the three MAP kinases and GSK3β are importantly all strong candidates as tau kinases that may be involved in the pathogenic hyperphosphorylation of tau in Alzheimer's disease.
Autoxidation and Neurotoxicity of 6‐Hydroxydopamine in the Presence of Some Antioxidants
Authors: Soto‐Otero Ramón , Méndez‐Álvarez Estefanía , Hermida‐Ameijeiras Álvaro , Muñoz‐Patiño Ana María , Labandeira‐Garcia José Luis
Published: March 2003

Abstract: 6‐Hydroxydopamine (6‐OHDA) is a dopaminergic neurotoxin
putatively involved in the pathogenesis of Parkinson's disease (PD). Its
neurotoxicity has been related to the production of reactive oxygen species.
In this study we examine the effects of the antioxidants ascorbic acid (AA),
glutathione (GSH), cysteine (CySH), and N‐acetyl‐CySH (NAC) on the
autoxidation and neurotoxicity of 6‐OHDA. In vitro, the autoxidation of 6‐OHDA
proceeds rapidly with the formation of H2O2 and with the
participation of the H2O2 produced in the reaction. The
presence of AA induced a reduction in the consumption of O2 during
the autoxidation of 6‐OHDA and a negligible presence of the
p‐quinone, which demonstrates the efficiency of AA to act as a redox
cycling agent. The presence of GSH, CySH, and NAC produced a significant
reduction in the autoxidation of 6‐OHDA. In vivo, the presence of sulfhydryl
antioxidants protected against neuronal degeneration in the striatum, which
was particularly remarkable in the case of CySH and was attributed to its
capacity to remove the H2O2 produced in the autoxidation of 6‐OHDA. These results corroborate the involvement of oxidative stress as the major mechanism in the neurotoxicity of 6‐OHDA and the putative role of CySH as a scavenger in relation to PD.
In Vitro Antioxidant Neuroprotective Activity of BN 80933, a Dual Inhibitor of Neuronal Nitric Oxide Synthase and Lipid Peroxidation
Authors: Demerlé‐Pallardy Caroline , Gillard‐Roubert Véronique , Marin Jean‐Grégoire , Auguet Michel , Chabrier Pierre‐Etienne
Published: March 2003

Abstract: BN 80933, a dual inhibitor of neuronal nitric oxide synthase and lipid peroxidation, prevents in vivo brain ischemic/reperfusion injury. In the present study, BN 80933 was shown to protect neurons from hypoxia‐induced cell death in primary cultures of cortical neurons. BN 80933 prevented lactate dehydrogenase activity elevation induced by hypoxia, displaying an IC50 value of 0.15 ± 0.05 μM. This effect was likely due to the antioxidant properties of BN 80933 because Trolox, but not NG‐nitro‐L‐arginine, also elicited protection. The antioxidant property of BN 80933 was then further investigated on HT‐22 cells subjected to buthionine sulfoximine‐ or glutamate‐induced glutathione depletion. The relative order of potency of the various compounds to inhibit oxidative stress‐induced neuronal death (BN 80933 > U104067 > butylated hydroxytoluene > 17β‐estradiol > Trolox > vitamin E) correlated with their ability to inhibit brain membrane lipid peroxidation (correlation coefficient = 0.939). BN 80933 afforded protection even when added 6 h after glutamate exposure. BN 80933 did not reverse intracellular glutathione depletion but prevented elevation of the level of 8‐epiprostaglandin F2α (8‐isoprostane), which appeared to be a delayed phenomenon. In conclusion, BN 80933 induces a potent cytoprotection that may be mediated by inhibition of delayed lipid peroxidation.
Analyses of Signal Transduction Cascades Reveal an Essential Role of Calcium Ions for Regulation of Melatonin Biosynthesis in the Light‐Sensitive Pineal Organ of the Rainbow Trout (Oncorhynchus mykiss)
Authors: Kroeber Susanne , Meissl Hilmar , Maronde Erik , Korf Horst‐Werner
Published: March 2003

Abstract: Signal transduction processes regulating melatonin production in the light‐sensitive trout pineal organ were investigated by immunocytochemical and immunochemical demonstration of phosphorylated cyclic AMP‐responsive element‐binding protein (pCREB) and measurements of cyclic AMP, melatonin, and calcium levels. Melatonin levels were tightly controlled by light and darkness. Elevation of cyclic AMP levels by 8‐bromo‐cyclic AMP, forskolin, and 3‐isobutyl‐1‐methylxanthine increased the levels of pCREB and melatonin in light‐ or dark‐adapted pineal organs in vitro. Without pharmacological treatment, the levels of pCREB and cyclic AMP remained constant for several hours before and after light onset. Inhibition of cyclic AMP‐dependent proteasomal proteolysis by lactacystin, MG 132, and calpain inhibitor I did not prevent the rapid, light‐induced suppression of melatonin biosynthesis. However, changes in the intracellular calcium concentration by drugs affecting voltagegated calcium channels of the L type and intracellular calcium oscillations (cobalt chloride, nifedipine, Bay K 8644) had dramatic effects on the rapid, light‐dependent changes in melatonin levels. These effects were not accompanied by changes in cyclic AMP levels. Thus, the rapid, light‐dependent changes in melatonin levels in the trout pineal organ are regulated apparently by a novel calcium signaling pathway and do not involve changes in cyclic AMP levels, cyclic AMP‐dependent proteasomal proteolysis, or phosphorylation of cyclic AMP‐responsive element‐binding protein.
Rod‐Type Transducin α‐Subunit Mediates a Phototransduction Pathway in the Chicken Pineal Gland
Authors: Kasahara Takaoki , Okano Toshiyuki , Yoshikawa Tomoko , Yamazaki Kazumitsu , Fukada Yoshitaka
Published: March 2003

Abstract : The chicken pineal gland is a photosensitive

neuroendocrine organ producing melatonin in circadian clock‐regulated and

light‐sensitive manners. To understand the relationship between the

photoreceptive molecule pinopsin and the light‐dependent melatonin suppression

that is sensitive to pertussis toxin treatment, we have searched for pertussis

toxin‐sensitive G protein α‐subunits expressed in the chicken pineal

gland. Here we report the cDNA cloning of the pineal transducin

α‐subunit (Gtα), which is highly homologous to human retinal rod

cell‐specific Gt1α. Concurrent cDNA cloning of chicken

retinal Gt1α and Gt2α (rod and cone

cell‐specific α‐subunits of transducin, respectively) revealed that the

chicken pineal Gtα is identical to the retinal Gt1α.

Double‐immunostaining analysis of the chicken pineal sections localized

Gt1α‐immunoreactivity in the rudimentary outer segments of

both follicular and parafollicular pinealocytes that were immunopositive to

anti‐pinopsin antibody. To examine whether pineal Gt1α is

involved in the pineal phototransduction pathway, trypsin protection assay was

applied for detecting the conversion of GDP‐bound Gt1α into

the guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS)‐bound

form in the pineal membrane homogenate. It was clearly demonstrated that the

pineal Gt1α is activated in a light‐dependent manner in the

presence of GTPγS. These data together suggest strongly that pineal

Gt1α mediates the phototransduction pathway triggered by pinopsin in the chicken pinealocytes.
Histamine Infusion Induces a Selective Dopaminergic Neuronal Death Along with an Inflammatory Reaction in Rat Substantia Nigra
Authors: Vizuete M L , Merino M , Venero J L , Santiago M , Cano J , Machado A
Published: March 2003

We have evaluated the effects of a direct infusion of histamine, as mediator of inflammatory response, in substantia nigra, striatum, medial septum, and medial lemniscus. Injection of 100 and 250 nmol of histamine in substantia nigra produced a selective damage in dopaminergic neurons evidenced by the loss of tyrosine hydroxylase mRNA‐expressing cells, tyrosine hydroxylase‐immunolabeled‐positive cell bodies, and dopamine and 3,4‐dihydroxyphenylacetic acid levels. In parallel we found an acute inflammatory response manifested by a loss of glial fibrillary acidic protein‐immunolabeled astrocytes and, at precisely the same area, an activation of microglia. In the striatum, only high doses (500 nmol) produced an evident terminal degeneration. The selective neurotoxicity of histamine for dopaminergic cells was demonstrated by the unaltered transcription of glutamic acid decarboxylase mRNA in substantia nigra. Moreover, intraseptal injection of 100 nmol of histamine failed to alter the pattern of choline acetyltransferase mRNA‐expressing cells, and intraparenchymal injection of histamine in medial lemniscus failed to alter the pattern of serotonin‐immunolabeled cells. We conclude that the substantia nigra is highly sensitive to histamine‐derived neurotoxicity, where inflammatory processes mediated by histamine could be important in the pathological changes that lead to dopaminergic neuronal damage after histamine infusion.
Astrocyte‐Derived Nitric Oxide Causes Both Reversible and Irreversible Damage to the Neuronal Mitochondrial Respiratory Chain
Authors: Stewart Victoria C , Sharpe Martyn A , Clark John B , Heales Simon J R
Published: March 2003

Cytokine‐stimulated astrocytes produce nitric oxide (NO), which, along with its metabolite peroxynitrite (ONOO‐), can inhibit components of the

mitochondrial respiratory chain. We used astrocytes as a source of

NO/ONOO‐ and monitored the effects on neurons in coculture. We

previously demonstrated that astrocytic NO/ONOO‐ causes significant damage to the activities of complexes II/III and IV of neighbouring neurons after a 24‐h coculture. Under these conditions, no neuronal death was observed. Using polytetrafluoroethane filters, which are permeable to gases such as NO but impermeable to NO derivatives, we have now demonstrated that astrocyte‐derived NO is responsible for the damage observed in our coculture system. Expanding on these observations, we have now shown that 24 h after removal of NO‐producing astrocytes, neurons exhibit complete recovery of complex II/III and IV activities. Furthermore, extending the period of exposure of neurons to NO‐producing astrocytes does not cause further damage to the neuronal mitochondrial respiratory chain. However, whereas the activity of complex II/III recovers with time, the damage to complex IV caused by a 48‐h coculture with NO‐producing astrocytes is irreversible. Therefore, it appears that neurons can recover from short‐term damage to mitochondrial complex II/III and IV, whereas exposure to astrocytic‐derived NO for longer periods causes permanent damage to neuronal complex IV.
Taurine Down‐Regulates Basal and Osmolarity‐Induced Gene Expression of Its Transporter, but Not the Gene Expression of Its Biosynthetic Enzymes, in Astrocyte Primary Cultures
Authors: Bitoun Marc , Tappaz Marcel
Published: March 2003

Abstract:Taurine content of astrocytes is primarily regulated by transport from the extracellular medium and endogenous biosynthesis from cysteine. We have investigated the gene expression of the taurine transporter (TauT) and the taurine biosynthetic enzymes, cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSD), in astrocyte primary cultures in relationship to cell taurine content. TauT, CDO, and CSD mRNA levels were determined through quantitative RT‐PCR. Cell taurine content was depleted by adapting the cells to a taurine‐free chemically defined medium and increased by incubating the cells in the same medium containing exogenous taurine. With increased cell taurine content the level of TauT mRNA decreased, whereas the levels of CDO and CSD mRNA remained unchanged. In astrocytes exposed to a hyperosmotic medium the TauT mRNA level increased, whereas the CDO and CSD mRNA levels were not significantly altered. The osmolarity‐induced up‐regulation of TauT mRNA expression was fully prevented by increasing cell taurine content. Thus, the gene expression of the taurine transporter, but not that of the taurine biosynthetic enzymes, appears to be under the control of two antagonistic regulations, namely, a taurine‐induced down‐regulation and an osmolarity‐induced up‐regulation.
Activation of Extracellular Signal‐Regulated Kinase (ERK) and Akt by Human Serotonin 5‐HT1B Receptors in Transfected BE(2)‐C Neuroblastoma Cells Is Inhibited by RGS4
Authors: Lione Angelique M. , Errico Monica , Lin Stanley L. , Cowen Daniel S.
Published: March 2003

Abstract: Regulator of G protein signaling (RGS) proteins are

GTPase‐activating proteins for heterotrimeric G proteins. One of the

best‐studied RGS proteins, RGS4, accelerates the rate of GTP hydrolysis by all

Gi and Gqα subunits yet has been shown to exhibit

receptor selectivity. Although RGS4 is expressed primarily in brain, its

effect on modulating the activity of serotonergic receptors has not yet been

reported. In the present study, transfected BE(2)‐C human neuroblastoma cells

expressing human 5‐HT1B receptors were used to demonstrate that

RGS4 can inhibit the coupling of 5‐HT1B receptors to cellular signals. Serotonin and sumatriptan were found to stimulate activation of extracellular signal‐regulated kinase. This activation was attenuated, but not completely inhibited, by RGS4. Similar inhibition by RGS4 of the protein kinase Akt was also observed. As RGS4 is expressed at high levels in brain, these results suggest that it may play a role in regulating serotonergic pathways.
Messenger RNAs Located in Myelin Sheath Assembly Sites
Authors: Gould Robert M. , Freund Concetta M. , Palmer Frank , Feinstein Douglas L.
Published: March 2003

Abstract: The targeting of mRNAs to specific subcellular locations is believed to facilitate the rapid and selective incorporation of their protein products into complexes that may include membrane organelles. In oligodendrocytes, mRNAs that encode myelin basic protein (MBP) and select myelin‐associated oligodendrocytic basic proteins (MOBPs) locate in myelin sheath assembly sites (MSAS). To identify additional mRNAs located in MSAS, we used a combination of subcellular fractionation and suppression subtractive hybridization. More than 50% of the 1,080 cDNAs that were analyzed were derived from MBP or MOBP mRNAs, confirming that the method selected mRNAs enriched in MSAS. Of 90 other cDNAs identified, most represent one or more mRNAs enriched in rat brain myelin. Five cDNAs, which encode known proteins, were characterized for mRNA size(s), enrichment in myelin, and tissue and developmental expression patterns. Two of these, peptidylarginine deiminase and ferritin heavy chain, have recognized roles in myelination. The corresponding mRNAs were of different sizes than the previously identified mRNA, and they had tissue and development expression patterns that were indistinguishable from those of MBP mRNA. Three other cDNAs recognize mRNAs whose proteins (SH3p13, KIF1A, and dynein light intermediate chain) are involved in membrane biogenesis. Although enriched in myelin, the tissue and developmental distribution patterns of these mRNAs differed from those of MBP mRNA. Six other cDNAs, which did not share significant sequence homology to known mRNAs, were also examined. The corresponding mRNAs were highly enriched in myelin, and four had tissue and developmental distribution patterns indistinguishable from those of MBP mRNA. These studies demonstrate that MSAS contain a diverse population of mRNAs, whose locally synthesized proteins are placed to contribute to myelin sheath assembly and maintenance. Characterization of these mRNAs and proteins will help provide a comprehensive picture of myelin sheath assembly.
δ Ca2+/Calmodulin‐Dependent Protein Kinase IIIsozyme‐Specific Induction of Neurite Outgrowth in P19 Embryonal Carcinoma Cells
Authors: Johnson Lesley D. , Willoughby Charlotte A. , Burke Sarah H. , Paik David S. , Jenkins Kimberly J. , Tombes Robert M.
Published: March 2003

Abstract: Ca2+/calmodulin‐dependent protein kinase II(CaMK‐II) has been linked to the induction of differentiation in preneuronalcells. In these cells, δ isozymes represent the majority of CaMK‐IIsexpressed and are activated by differentiation stimuli. To determine whetherδ CaMK‐IIs are causative or coincident with in vitro differentiation, weoverexpressed wild‐type, constitutively active, and C‐terminal domains ofδ and γ CaMK‐II isozymes in mouse P19 and NIH/3T3 cells usinghigh‐efficiency transfections. At 1‐2 days after transfection, onlyconstitutively active δ CaMK‐II isozymes induced branched cellularextensions in both cell types. In P19 cells, retinoic acid induced neuriteextensions after 3‐4 days; these extensions were coincident with a fourfoldincrease in endogenous CaMK‐II activity. Extensions induced by both retinoicacid and δ CaMK‐IIs contained class III β‐tubulin in adiscontinuous or beaded pattern. C‐terminal CaMK‐II constructs disrupted theability of endogenous CaMK‐II to autophosphorylate and blocked retinoicacid‐induced differentiation. δ CaMK‐II was found along extensions,whereas γ CaMK‐II exhibited a more diffuse, cytosolic localization.These data not only support an extranuclear role for CaMK‐II in promotingneurite outgrowth, but also demonstrate CaMK‐II isozyme specificity in theseearly steps of neuronal differentiation.
Cyclic GMP/Cyclic GMP‐Dependent Protein Kinase System Prevents Excitotoxicity in an Immortalized Oligodendroglial Cell Line
Authors: Yoshioka Akira , Yamaya Yoko , Saiki Shinji , Kanemoto Masumi , Hirose Genjiro , Pleasure David
Published: March 2003

Abstract: Previously, we have demonstrated that excitotoxicity of oligodendrocyte‐like cells (OLC), differentiated from immortalized rat O‐2A progenitor cells (CG‐4 cells), is prevented by cyclic AMP‐elevating agents. We now report that some agents that elevate cyclic GMP prevent OLC excitotoxicity. Kainate‐induced injury was prevented by cyclic GMP analogues (8‐bromo‐cyclic GMP and dibutyryl cyclic GMP), a guanylate cyclase activator [atrial natriuretic peptide (ANP)], and phosphodiesterase inhibitors [3‐isobutyl‐1‐methylxanthine (IBMX), ibudilast, propentofylline, and rolipram]. When both forskolin and 8‐bromo‐cyclic GMP were added, kainate‐induced injury was additively prevented. There was a strong positive correlation between suppression of kainate‐induced Ca2+ influx and prevention of injury by these chemicals. The measurement of intracellular cyclic AMP and cyclic GMP by radioimmunoassay demonstrated the following: an increase of cyclic GMP with treatment with 8‐bromo‐cyclic GMP, dibutyryl cyclic GMP, and ANP; an increase of cyclic AMP with treatment with ibudilast and rolipram; and an increase of both cyclic AMP and cyclic GMP with treatment with IBMX and propentofylline. Kainate‐induced Ca2+ influx was decreased by 8‐(4‐chlorophenylthiol)‐guanosine‐3′,5′‐monophosphate, an activator of cyclic GMP‐dependent protein kinase (PKG), or okadaic acid, an inhibitor of protein phosphatases 1 and 2A. RT‐PCR and western blotting of OLC demonstrated transcription of PKG II gene and translation of PKG Iβ mRNA, but no translation of PKG Iα mRNA. Therefore, we concluded that the cyclic GMP/PKG system prevents OLC excitotoxicity.
Effect of Amphotericin B on Wild‐Type and Mutated Prion Proteins in Cultured Cells
Authors: Mangé Alain , Milhavet Ollivier , McMahon Hilary E. M. , Casanova Danielle , Lehmann Sylvain
Published: March 2003

Abstract: Transmissible spongiform encephalopathies form a group of fatal neurodegenerative disorders that have the unique property of being infectious, sporadic, or genetic in origin. Although some doubts remain on the nature of the responsible agent of these diseases, it is clear that a protein called PrPSc [the scrapie isoform of prion protein (PrP)] plays a central role. PrPSc represents a conformational variant of PrPC (the cellular isoform of PrP), the normal host protein. Polyene antibiotics, such as amphotericin B, have been shown to delay the accumulation of PrPSc and to increase the incubation time of the disease after experimental transmission in laboratory animals. Unlike agents such as Congo red, the inhibitory effect of amphotericin B on PrPSc generation has not been observed in infected cultures. Using transfected cells expressing wild‐type or mutated mouse PrPs, we show here that amphotericin B is able to interfere with the generation of abnormal PrP isoforms in culture. Its action seems related to a modification of PrP trafficking through the association of this glycosylphosphatidylinositol‐anchored protein with detergent‐resistant microdomains. These results represent a first step toward the comprehension of the mechanism of action of amphotericin B in transmissible spongiform encephalopathies.
Pathways of Neurosteroid Biosynthesis in Cell Lines from Human Brain
Authors: Brown Rachel C. , Cascio Caterina , Papadopoulos Vassilios
Published: March 2003

Abstract: Neurosteroids in rodents can originate from peripheral tissues or be locally synthesized in specific brain areas. There is, as yet, no information about the synthesis and regulation of neurosteroids in human brain. We examined the ability of human brain cells to synthesize steroids from a radiolabeled precursor and the mRNA and protein expression of key components of peripheral steroidogenic machinery. Oligodendrocytes are the source of pregnenolone in human brain. Human astrocytes do not synthesize radiolabeled pregnenolone, nor do human neurons. There is potential for all three cell types to metabolize pregnenolone to other neurosteroids, including dehydroepiandrosterone. mRNA and protein for cytochrome P450 17α‐hydroxylase were found in all cell types, although no activity could be demonstrated. We examined the ability of the cells to make dehydroepiandrosterone via an alternative pathway induced by treatment with Fe2+. Oligodendrocytes and astrocytes make dehydroepiandrosterone via this pathway, but neurons do not. In searching for a natural regulator of dehydroepiandrosterone formation, we observed that treating oligodendrocytes with β‐amyloid, which increases reactive oxygen species, also increased dehydroepiandrosterone formation. These effects of β‐amyloid were blocked by vitamin E. These results indicate that human brain makes steroids in a cell‐specific manner and suggest that dehydroepiandrosterone synthesis can be regulated by intracellular free radicals.
Complementation of Physiological and Behavioral Defects by a Slowpoke Ca2+ ‐Activated K+ Channel Transgene
Authors: Brenner Robert , Yu Joyce Y. , Srinivasan Karpagam , Brewer Lawrence , Larimer James L. , Wilbur Jennette L. , Atkinson Nigel S.
Published: March 2003

Abstract: The Drosophila slowpoke gene encodes a large conductance calcium‐activated potassium channel used in neurons, muscle, and some epithelial cells. Tissue‐specific transcriptional promoters and alternative mRNA splicing generate a large array of transcripts. These distinct transcripts are thought to tailor the properties of the channel to the requirements of the cell. Presumably, a single splice variant cannot satisfy the specific needs of all cell types. To test this, we examined whether a single slowpoke splice variant was capable of complementing all slowpoke behavioral phenotypes. Null mutations in slowpoke cause animals to be semiflightless and to manifest an inducible "sticky‐feet" phenotype. The well‐characterized slowpoke transcriptional control region was used to direct the expression of a single slowpoke splice variant (cDNA H13) in transgenic flies. The endogenous gene in these flies had been inactivated by the slo4 mutation. Action‐potential recordings and voltage‐clamp recordings demonstrated the production of functional channels from the transgene. The transgene completely complemented the flight defect, but not the sticky‐feet phenotype. We conclude that distinct slowpoke channel isoforms, produced by alternative splicing, are not interchangeable and are required for proper function of different cell types.
Potentiation by histamine of synaptically mediated excitotoxicity in cultured hippocampal neurones: a possible role for mast cells
Authors: Skaper Stephen D. , Facci Laura , Kee Wai Jing , Strijbos Paul J. L. M.
Published: March 2003

Excessive glutamatergic neurotransmission, particularly when mediated by the N‐methyl‐d‐aspartate (NMDA) subtype of glutamate receptor, is thought to underlie neuronal death in a number of neurological disorders. Histamine has been reported to potentiate NMDA receptor‐mediated events under a variety of conditions. In the present study we have utilized primary hippocampal neurone cultures to investigate the effect of mast cell‐derived, as well as exogenously applied, histamine on neurotoxicity evoked by excessive synaptic activity. Exposure of mature cultures for 15 min to an Mg2+‐free/glycine‐containing buffer to trigger synaptic transmission through NMDA receptors, caused a 30–35% neuronal loss over 24 h. When co‐cultured with hippocampal neurones, activated mast cells increased excitotoxic injury to 60%, an effect that was abolished in the presence of histaminase. Similarly, addition of histamine during magnesium deprivation produced a concentration‐dependent potentiation (+ 60%; EC50 : 5 µm) of neuronal death which was inhibited by sodium channel blockers and NMDA receptor antagonists, although this effect did not involve known histamine receptors. The histamine effect was further potentiated by acidification of the culture medium. Cultures ‘preconditioned’ by sublethal (5 min) Mg2+ deprivation exhibited less neuronal death than controls when exposed to a more severe insult. NMDA receptor activation and the extracellular regulated kinase cascade were required for preconditioning neuroprotection. The finding that histamine potentiates NMDA receptor‐mediated excitotoxicity may have important implications for our understanding of conditions where enhanced glutamatergic neurotransmission is observed in conjunction with tissue acidification, such as cerebral ischaemia and epilepsy.
A bioreversible prodrug approach designed to shift mechanism of brain uptake for amino‐acid‐containing anticancer agents
Authors: Killian Dennis M. , Chikhale Prashant J.
Published: March 2003

By derivatization at the N‐terminus of amino acid‐based anticancer agents (e.g. melphalan and acivicin) to form a drug delivery system (TDDS), we demonstrate a change in the mechanism of brain uptake from the large neutral amino acid transporter (LAT) pathway to passive. An in situ rat brain perfusion technique was used to determine the brain capillary permeability–surface area (PA) product for [14C]l‐Leu as control (5.18 ± 0.32 × 10−2 mL/s/g), which was inhibited competitively (to 7–18% of control) by an excess concentration of the amino‐acid‐containing anticancer agents, acivicin and melphalan. However, TDDS did not compete for LAT‐mediated brain uptake of the radiotracer [14C]l‐Leu. Brain uptake of TDDS was determined after in situ brain perfusion followed by RP‐HPLC along with LC‐MS/MS detection of the analytes in brain samples. The PA product for CH3‐TDDS containing melphalan (5.09 ± 2.0 × 10−2 mL/s/g) shows that these agents rapidly cross the blood–brain barrier. Furthermore, competition studies of CH3‐TDDS with [3H]verapamil suggest that the TDDS interacts significantly with the multidrug resistant efflux system (P‐glycoprotein) at the blood–brain barrier. Therefore, TDDS were shown to lack LAT‐mediated brain uptake. The drug delivery systems, however, showed uptake predominantly via the passive route along with recognition by the multidrug resistant efflux protein at the cerebrovasculature.
On the mechanisms of neuroprotection by creatine and phosphocreatine
Authors: Brustovetsky Nickolay , Brustovetsky Tatiana , Dubinsky Janet M.
Published: March 2003

Creatine and phosphocreatine were evaluated for their ability to prevent death of cultured striatal and hippocampal neurons exposed to either glutamate or 3‐nitropropionic acid (3NP) and to inhibit the mitochondrial permeability transition in CNS mitochondria. Phosphocreatine (PCr), and to a lesser extent creatine (Cr), but not (5R,10S)‐(+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine hydrogen maleate (MK801), dose‐dependently ameliorated 3NP toxicity when applied simultaneously with the 3NP in Mg2+‐free media. Pre‐treatment of PCr for 2 or 5 days and Cr for 5 days protected against glutamate excitotoxicity equivalent to that achieved by MK801 post‐treatment. The combination of PCr or Cr pre‐treatment and MK801 post‐treatment did not provide additional protection, indicating that both prevented the toxicity attributable to secondary glutamate release. To determine if Cr or PCr directly inhibited the permeability transition, mitochondrial swelling and depolarization were assayed in isolated, purified brain mitochondria. PCr reduced the amount of swelling induced by calcium by 20%. Cr decreased mitochondrial swelling when inhibitors of creatine kinase octamer–dimer transition were present. However, in brain mitochondria prepared from rats fed a diet supplemented with 2% creatine for 2 weeks, the extent of calcium‐induced mitochondrial swelling was not altered. Thus, the neuroprotective properties of PCr and Cr may reflect enhancement of cytoplasmic high‐energy phosphates but not permeability transition inhibition.
Neuronal death is an active, caspase‐dependent process after moderate but not severe DNA damage
Authors: Gobbel Glenn T. , Chan Pak H.
Published: March 2003

Mild insults to neurons caused by ischemia or glutamate induce apoptosis, whereas severe insults induce non apoptotic death, such as necrosis. The molecular targets that are damaged by these insults and ultimately induce cell death are not fully established. To determine if DNA damage can induce apoptotic or non apoptotic death depending on the severity, neurons were treated with up to 128 Gy of ionizing radiation. Such treatment induced a dose‐related increase in DNA single‐strand breaks but no immediate membrane disruption or lipid peroxidation. Following moderate doses of  32 Gy, neuronal death had many characteristics of apoptosis including nuclear fragmentation and DNA laddering. Nuclear fragmentation and membrane breakdown after moderate DNA damage could be blocked by inhibition of active protein synthesis with cycloheximide and by inhibition of caspases. In contrast, cell death after doses of > 32 Gy was not blocked by cycloheximide or caspase inhibitors, and membrane breakdown occurred relatively early in the cell death process. These data suggest that cell death after high dose irradiation and severe DNA damage can occur by non apoptotic mechanisms and that blocking apoptotic pathways may not prevent death after severe damage.
Phosphorylation state of the native high‐molecular‐weight neurofilament subunit protein from cervical spinal cord in sporadic amyotrophic lateral sclerosis
Authors: Strong Michael J. , Strong Wendy L. , Jaffe Howard , Traggert Bearnd , Sopper Maggie M. , Pant Harish C.
Published: March 2003

The intraneuronal aggregation of phosphorylated high‐molecular‐weight neurofilament protein (NFH) in spinal cord motor neurons is considered to be a key pathological marker of amyotrophic lateral sclerosis (ALS). In order to determine whether this observation is due to the aberrant or hyper‐phosphorylation of NFH, we have purified and characterized NFH from the cervical spinal cords of ALS patients and controls. We observed no differences between ALS and normal controls in the physicochemical properties of NFH in Triton X‐100 insoluble protein fractions, with respect to migration patterns on 2D‐iso electrofocusing (IEF) gels, the rate of Escherichia coli alkaline phosphatase mediated dephosphorylation, or the rate of calpain‐mediated proteolysis. The rate of calpain‐mediated proteolysis was unaffected by either exhaustive NFH dephosphorylation or by the addition of calmodulin to the reaction. Phosphopeptides and the phosphorylated motifs characterized by liquid chromatography tandem mass spectroscopy (LC/MS/MS) analysis demonstrated that all the phosphorylated residues found in ALS NFH were also found to be phosphorylated in normal human NFH samples. Hence, we have observed no difference in the physicochemical properties of normal and ALS NFH extracted from cervical spinal cords, suggesting that the perikaryal aggregation of highly phosphorylated NF in ALS neurons reflects the aberrant somatotopic localization of normally phosphorylated NFH.
Dopaminergic cell death induced by MPP+, oxidant and specific neurotoxicants shares the common molecular mechanism
Authors: . Chun Hong S , Gibson Gary E. , DeGiorgio Lorraine A. , Zhang H. , Kidd Vincent J. , Son Jin H.
Published: March 2003

Recent etiological study in twins (Tanneret al. 1999) strongly suggests that environmental factors play an important role in typical, non‐familial Parkinson's disease (PD), beginning after age 50. Epidemiological risk factor analyses of typical PD cases have identified several neurotoxicants, including MPP+(the active metabolite of MPTP), paraquat, dieldrin, manganese and salsolinol. Here, we tested the hypothesis that these neurotoxic agents might induce cell death in our nigral dopaminergic cell line, SN4741 (Sonet al. 1999) through a common molecular mechanism. Our initial experiments revealed that treatment with both MPP+and the other PD‐related neurotoxicants induced apoptotic cell death in SN4741 cells, following initial increases of H2O2‐related ROS activity and subsequent activation of JNK1/2 MAP kinases. Moreover, we have demonstrated that during dopaminergic cell death cascades, MPP+, the neurotoxicants and an oxidant, H2O2equally induce the ROS‐dependent events. Remarkably, the oxidant treatment alone induced similar sequential molecular events: ROS increase, activation of JNK MAP kinases, activation of the PITSLRE kinase, p110, by both Caspase‐1 and Caspase‐3‐like activities and apoptotic cell death. Pharmacological intervention using the combination of the antioxidant Trolox and a pan‐caspase inhibitor Boc‐(Asp)‐fmk (BAF) exerted significant neuroprotection against ROS‐induced dopaminergic cell death. Finally, the high throughput cDNA microarray screening using the current model identified downstream response genes, such as heme oxygenase‐1, a constituent of Lewy bodies, that can be the useful biomarkers to monitor the pathological conditions of dopaminergic neurons under neurotoxic insult.
Inhibition of tumour necrosis factor‐α (TNFα)‐induced NF‐κB p52 converts the metabolic effects of microglial‐derived TNFα on mouse cerebellar neurones to neurotoxicity
Authors: Nicholas Richard St J. , Compston Alastair , Brown David R.
Published: March 2003

Activated microglia are implicated in the injury of neurones and macroglia bothin vitroandin vivo.Here, we demonstrate that media conditioned by interferon‐γ treated microglia initially impair the metabolism of mouse cerebellar neurones grown in serum‐free conditions without inducing cell death. Metabolic effects include inhibition of the ability of mitochondria to reduce 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide (MTT) and cytochrome oxidase activity. These effects are blocked by antibodies to tumour necrosis factor‐α (TNFα), a cytokine produced by microglial activation, and they are not reproduced by media conditioned by resting microglia. The metabolic effects are evident for up to 24 hin vitro. More prolonged exposure, up to 48 h, results in TNFα dependent neuronal death as previously observed. Between 2 and 48 h TNFα present in media conditioned by interferon‐γ treated but not resting microglia is associated with nuclear factor kappa B (NF‐κB) consensus sequence binding in paired mouse cerebellar neuronal cultures without affecting activation of the signal transducer and activator of transcription (STAT) transcription factor. Neuronal death can be accelerated by peptide blockade of the nuclear transport of NF‐κB p52 subunit during exposure of cerebellar neurones to medium from interferon‐γ treated microglia. This toxicity is blocked by anti‐TNFα antibody. Soluble factors released by activated microglia therefore contribute to neuronal dysfunction that is initially reversible but may culminate in neurotoxicity. Characterizing and manipulating these eventsin vivotheoretically provides an opportunity for neuroprotection in selected diseases affecting the central nervous system.
Sphingosine kinase expression regulates apoptosis and caspase activation in PC12 cells
Authors: Edsall Lisa C. , Cuvillier Olivier , Twitty Sharon , Spiegel Sarah , Milstien Sheldon
Published: March 2003

Sphingosine‐1‐phosphate (SPP), a bioactive sphingolipid metabolite, suppresses apoptosis of many types of cells, including rat pheochromocytoma PC12 cells. Elucidating the molecular mechanism of action of SPP is complicated by many factors, including uptake and metabolism, as well as activation of specific G‐protein‐coupled SPP receptors, known as the endothelial differentiation gene‐1 (EDG‐1) family. In this study, we overexpressed type 1 sphingosine kinase (SPHK1), the enzyme that converts sphingosine to SPP, in order to examine more directly the role of intracellularly generated SPP in neuronal survival. Enforced expression of SPHK1 in PC12 cells resulted in significant increases in kinase activity, with corresponding increases in intracellular SPP levels and concomitant decreases in both sphingosine and ceramide, and marked suppression of apoptosis induced by trophic factor withdrawal or by C2‐ceramide. NGF, which protects PC12 cells from serum withdrawal‐induced apoptosis, also stimulated SPHK1 activity. Surprisingly, overexpression of SPHK1 had no effect on activation of two known NGF‐stimulated survival pathways, extracellular signal regulated kinase ERK 1/2 and Akt. However, trophic withdrawal‐induced activation of the stress activated protein kinase, c‐Jun amino terminal kinase (SAPK/JNK), and activation of the executionary caspases 2, 3 and 7, were markedly suppressed. Moreover, this abrogation of caspase activation, which was prevented by the SPHK inhibitor N,N‐dimethylsphingosine, was not affected by pertussis toxin treatment, indicating that the cytoprotective effect was likely not mediated by binding of SPP to cell surface Gi‐coupled SPP receptors. In agreement, there was no detectable release of SPP into the culture medium, even after substantially increasing cellular SPP levels by NGF or sphingosine treatment. In contrast to PC12 cells, C6 astroglioma cells secreted SPP, suggesting that SPP might be one of a multitude of known neurotrophic factors produced and secreted by glial cells. Collectively, our results indicate that SPHK/SPP may play an important role in neuronal survival by regulating activation of SAPKs and caspases.
Rescue from death but not from functional impairment: caspase inhibition protects dopaminergic cells against 6‐hydroxydopamine‐induced apoptosis but not against the loss of their terminals
Authors: Coelln Rainer von , Kügler Sebastian , Bähr Mathias , Weller Michael , Dichgans Johannes , Schulz Jörg B.
Published: March 2003

Despite the identification of several mutations in familial Parkinson's disease (PD), the underlying mechanisms of dopaminergic neuronal loss in idiopathic PD are still unknown. To study whether caspase‐dependent apoptosis may play a role in the pathogenesis of PD, we examined 6‐hydroxydopamine (6‐OHDA) toxicity in dopaminergic SH‐SY5Y cells and in embryonic dopaminergic mesencephalic cultures. 6‐OHDA induced activation of caspases 3, 6 and 9, chromatin condensation and cell death in SH‐SY5Y cells. The caspase inhibitor benzyloxycarbonyl‐Val‐Ala‐Asp‐(O‐methyl)fluoromethylketone (zVAD‐fmk) or adenovirally mediated ectopic expression of the X‐chromosomal inhibitor of apoptosis protein (XIAP) blocked caspase activation and prevented death of SH‐SY5Y cells. Similarly, zVAD‐fmk provided protection from 6‐OHDA‐induced loss of tyrosine hydroxylase‐positive neurones in mesencephalic cultures. In contrast, zVAD‐fmk failed to protect mesencephalic dopaminergic neurones from 6‐OHDA‐induced loss of neurites and reduction of [3H]dopamine uptake. These data suggest that, although caspase inhibition provides protection from 6‐OHDA‐induced death of dopaminergic neurones, the neurones may remain functionally impaired.