bluuebunny/crossref_metadata_2025 huggingface dataset:
Pilocarpine‐Induced Status Epilepticus Causes N‐Methyl‐D‐Aspartate Receptor‐Dependent Inhibition of Microsomal Mg2+/Ca2+ ATPase‐Mediated Ca2+ Uptake
Authors: Parsons J. Travis , Churn Severn B. , Kochan Lisa D. , DeLorenzo Robert J.
Published: March 2003

Abstract: Status epilepticus is associated with sustained and

elevated levels of cytosolic Ca2+. To elucidate the mechanisms

associated with changes of cytosolic Ca2+ after status epilepticus,

this study was initiated to evaluate the effect of pilocarpine‐induced status

epilepticus on Mg2+/Ca2+ ATPase‐mediated Ca2+

uptake in microsomes isolated from rat cortex, because the Ca2+

uptake mechanism plays a major role in regulating intracellular

Ca2+ levels. The data demonstrated that the initial rate and

overall Ca2+ uptake in microsomes from pilocarpinetreated animals

were significantly inhibited compared with those in microsomes from

saline‐treated control animals. It was also shown that the inhibition of

Ca2+ uptake caused by status epilepticus was not an artifact of

increased Ca2+ release from microsomes, selective isolation of

damaged microsomes from the homogenate, or decreased

Mg2+/Ca2+ ATPase protein in the microsomes. Pretreatment

with the NMDA antagonist dizocilpine maleate blocked status

epilepticus‐induced inhibition of the initial rate and overall Ca2+

uptake. The data suggest that inhibition of microsomal

Mg2+/Ca2+ ATPase Ca2+ uptake is involved in

NMDA‐dependent deregulation of cytosolic Ca2+ homeostasis associated with status epilepticus.
Two Conserved Arginines in the Extracellular N‐Terminal Domain of the GABAA Receptor α5 Subunit Are Crucial for Receptor Function
Authors: Hartvig Line , Lükensmejer Birthe , Liljefors Tommy , Dekermendjian Kim
Published: March 2003

Abstract: The γ‐aminobutyric acid (GABA) binding pocket within
the GABAA receptor complex has been suggested to contain arginine
residues. The aim of this study was to test this hypothesis by mutating
arginine residues potentially contributing to the formation of a GABA binding
pocket. Thus, six arginines conserved in human GABAA receptor
α subunits (arginine 34, 70, 77, 123, 135, and 224) as well as two
nonconserved arginines (79 and 190), all located in the extracellular
N‐terminal segment of the α5 subunit, were substituted by
lysines. The individual α5 subunit mutants were coexpressed
with human β2 and γ2s GABAA
receptor subunits in Chinese hamster ovary cells by transient transfection.
Electrophysiological whole‐cell patch‐clamp recordings show that, of the eight
arginine residues tested, the two arginines at positions 70 and 123 appear to
be essential for the GABA‐gated chloride current because the EC50
values of the two mutant constructs increase >100‐fold compared with the
wild‐type α5,β2,γ2s
GABAA receptor. However, diazepam and allopregnanolone modulation and pentobarbital stimulation properties are unaffected by the introduction of lysines at positions 70 and 123. A double mutant carrying lysine substitutions at positions 70 and 123 is virtually insensitive to GABA, suggesting alterations of one or more GABA binding sites.
Impaired Mitochondrial Function Results in Increased Tissue Transglutaminase Activity In Situ
Authors: Lesort Mathieu , Tucholski Janusz , Zhang Jianwen , Johnson Gail V. W.
Published: March 2003

Abstract: Tissue transglutaminase (tTG) is a transamidating enzyme that is elevated in Huntington's disease (HD) brain and may be involved in the etiology of the disease. Further, there is evidence of impaired mitochondrial function in HD. Therefore, in this study, we examined the effects of mitochondrial dysfunction on the transamidating activity of tTG. Neuroblastoma SH‐SY5Y cells stably overexpressing human tTG or mutated inactive tTG were treated with 3‐nitropropionic acid (3‐NP), an irreversible inhibitor of succinate dehydrogenase. 3‐NP treatment of tTG‐expressing cells resulted in a significant increase of TG activity in situ. In vitro measurements demonstrated that 3‐NP had no direct effect on tTG activity. However, 3‐NP treatment resulted in a significant decrease of the levels of GTP and ATP, two potent inhibitors of the transamidating activity of tTG. No significant changes in the intracellular levels of calcium were observed in 3‐NP‐treated cells. Treatment with 3‐NP in combination with antioxidants significantly reduced the 3‐NP‐induced increase in in situ TG activity, demonstrating that oxidative stress is a contributing factor to the increase of TG activity. This study demonstrates for the first time that impairment of mitochondrial function significantly increases TG activity in situ, a finding that may have important relevance to the etiology of HD.
Reversible Inactivation of Superoxide‐Sensitive Aconitase in Aβ1‐42‐Treated Neuronal Cell Lines
Authors: Longo Valter D. , Viola Kirsten L. , Klein William L. , Finch Caleb E.
Published: March 2003

Abstract: The activity of the superoxide‐sensitive enzyme aconitase was monitored to evaluate the generation of superoxide in neuronal cell lines treated with β‐amyloid (Aβ) peptide 1‐42. Treatment of differentiated and undifferentiated rat PC12 and human neuroblastoma SK‐N‐SH cells with soluble Aβ1‐42 (Aβ‐derived diffusible ligands) or fibrillar Aβ1‐42 caused a 35% reversible inactivation of aconitase, which preceded loss of viability and was correlated with altered cellular function. Aconitase was reactivated upon incubation of cellular extracts with iron and sulfur, suggesting that Aβ causes the release of iron from 4Fe‐4S clusters. Aβ neurotoxicity was partially blocked by the iron chelator deferoxamine. These data suggest that increased superoxide generation and the release of iron from 4Fe‐4S clusters are early events in Aβ1‐42 neurotoxicity.
Mice Transgenic for the Huntington's Disease Mutation Are Resistant to Chronic 3‐Nitropropionic Acid‐Induced Striatal Toxicity
Authors: Hickey Miriam A. , Morton A. Jennifer
Published: March 2003

Abstract: Neuronal loss in Huntington's disease (HD) is seen first in the neostriatum. It has been suggested that impaired metabolism underlies this degeneration, as striatal vulnerability to excitotoxicity is increased by metabolic compromise. At 12 weeks of age, a transgenic mouse carrying the HD mutation (R6/2 line) has been shown to have an increased vulnerability to the mitochondrial toxin 3‐nitropropionic acid (3‐NP). However, in contrast, younger R6/2 mice appear to be less vulnerable than wild‐type (WT) mice to the excitotoxins kainic acid and quinolinic acid (QA). In this study, we examine the possibility that the sensitivity of R6/2 mice to 3‐NP might be age dependent. We treated young, symptomatic R6/2 mice with 3‐NP and found that despite their progressive neurological phenotype, they were not more susceptible to 3‐NP intoxication than their WT littermates. Further, fewer R6/2 than WT mice developed striatal lesions. We suggest that compensatory mechanisms exist in the R6/2 mouse brain that protect it against the toxic effect of the transgene and coincidentally protect against exogenous toxins such as 3‐NP, QA, and kainic acid. The existence of similar compensatory mechanisms may explain why, in humans, HD is a late‐onset disorder, despite early expression of the genetic mutation.
Opposing Actions of Phosphatidylinositol 3‐Kinase and Glycogen Synthase Kinase‐3β in the Regulation of HSF‐1 Activity
Authors: Bijur Gautam N. , Jope Richard S.
Published: March 2003

Abstract: Elevated temperatures activate the survival promoters Aktand heat shock factor‐1 (HSF‐1), a transcription factor that induces theexpression of heat shock proteins (HSPs), such as HSP‐70. Because neuronalmechanisms controlling these responses are not known, these were investigatedin human neuroblastoma SH‐SY5Y cells. Heat shock (45°C) rapidly activatedAkt, extracellular signal‐regulated kinases 1 and 2 (ERK1/2), and p38, butonly Akt was activated in a phosphatidylinositol 3‐kinase (PI‐3K)‐dependentmanner, as the PI‐3K inhibitors LY294002 and wortmannin blocked Aktactivation, but not ERK1/2 or p38 activation. Akt activation was not blockedby inhibition of p38 or ERK1/2, indicating the independence of these signalingsystems. Heat shock treatment also caused a rapid increase in HSF‐1 DNAbinding activity that was partially dependent on PI‐3K activity, as both thePI‐3K inhibitors attenuated this response. Because Akt inhibits glycogensynthase kinase‐3β (GSK‐3β), an enzyme that facilitates cell death,we tested if GSK‐3β is a negative regulator of HSF‐1 activation.Overexpression of GSK‐3β impaired heat shock‐induced activation of HSF‐1,and also reduced HSP‐70 production, which was partially restored by theGSK‐3β inhibitor lithium. Thus, heat shock‐induced activation of PI‐3Kand the inhibitory effect of GSK‐3β on HSF‐1 activation and HSP‐70expression imply that Akt‐induced inhibition of GSK‐3β contributes to theactivation of HSF‐1.
A 14‐3‐3 mRNA Is Up‐Regulated in Amyotrophic Lateral Sclerosis Spinal Cord
Authors: Malaspina Andrea , Kaushik Narendra , Belleroche Jackie de
Published: March 2003

Abstract: We have recently isolated a 2.2‐kb cDNA clone (1C5) from ahuman spinal cord cDNA library with partial identity to the 14‐3‐3 proteinmRNA encoding the θ protein (YWHAQ). 14‐3‐3 protein transcripts arehighly expressed in large projection neurones of the hippocampus, cerebellum,and spinal cord and have been found to be significantly up‐regulated in ratmotor neurones following hypoglossal nerve axotomy. In this study weinvestigated whether the 1C5 transcript (YWHAQ) isolated from spinal cord wasinvolved in amyotrophic lateral sclerosis (ALS). We found a significantup‐regulation of 1C5 (YWHAQ) in lumbar spinal cord from patients with sporadicALS compared with controls, with the highest levels of expression being foundin individuals with predominant lower motor neurone involvement. A 6‐bp tandemrepeat in the 5′‐untranslated region of the gene was found to bepolymorphic, but no significant association with disease was found followinggenomic analysis of this region. The localisation of 1C5 (YWHAQ) to chromosome2 was determined and coincides with that reported for clone HS1 (EMBLaccession no. X57347). These results show the marked up‐regulation of the14‐3‐3 isoform (YWHAQ) in ALS spinal cord and indicate the involvement of apotential 14‐3‐3‐mediated survival pathway in the pathogenesis of ALS.
α‐Synuclein Up‐Regulation in Substantia Nigra Dopaminergic Neurons Following Administration of the Parkinsonian Toxin MPTP
Authors: Vila Miquel , Vukosavic Slobodanka , Jackson‐Lewis Vernice , Neystat Michael , Jakowec Michael , Przedborski Serge
Published: March 2003

Abstract: Mutations in α‐synuclein cause a form of familial Parkinson’s disease (PD), and wild‐type α‐synuclein is a major component of the intraneuronal inclusions called Lewy bodies, a pathological hallmark of PD. These observations suggest a pathogenic role for α‐synuclein in PD. Thus far, however, little is known about the importance of α‐synuclein in the nigral dopaminergic pathway in either normal or pathological situations. Herein, we studied this question by assessing the expression of synuclein‐1, the rodent homologue of human α‐synuclein, in both normal and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐intoxicated mice. In normal mice, detectable levels of synuclein mRNA and protein were seen in all brain regions studied and especially in ventral midbrain. In the latter, there was a dense synuclein‐positive nerve fiber network, which predominated over the substantia nigra, and only few scattered synuclein‐positive neurons. After a regimen of MPTP that kills dopaminergic neurons by apoptosis, synuclein mRNA and protein levels were increased significantly in midbrain extracts; the time course of these changes paralleled that of MPTP‐induced dopaminergic neurodegeneration. In these MPTP‐injected mice, there was also a dramatic increase in the number of synuclein‐immunoreactive neurons exclusively in the substantia nigra pars compacta; all synuclein‐positive neurons were tyrosine hydroxylase‐positive, but none coexpressed apoptotic features. These data indicate that synuclein is highly expressed in the nigrostriatal pathway of normal mice and that it is up‐regulated following MPTP‐induced injury. In light of the synuclein alterations, it can be suggested that, by targeting this protein, one may modulate MPTP neurotoxicity and, consequently, open new therapeutic avenues for PD.
Involvement of Nitric Oxide in Pentylenetetrazole‐Induced Kindling in Rats
Authors: Han Daiken , Yamada Kiyofumi , Senzaki Kouji , Xiong Huabao , Nawa Hiroyuki , Nabeshima Toshitaka
Published: March 2003

Abstract: We investigated the role of nitric oxide (NO) and brain‐derived neurotrophic factor (BDNF) in the pentylenetetrazole (PTZ)‐induced kindling in rats. Seizures were induced by single administration of PTZ, which was associated with an increase in levels of NO metabolites (NOx) in the hippocampus. Pretreatment with a neuronal NO synthase inhibitor, 7‐nitroindazole (7‐NI), diminished the PTZ‐induced increase in NOx levels without affecting the seizure intensity. Repeated administration of PTZ produced a gradual increase in the seizure intensity, leading to the development of kindling. In the kindled rats, PTZ at a dose of 40 mg/kg increased NOx levels in the hippocampus, whereas it had no effect in control animals. Cotreatment of 7‐NI with PTZ blocked the development of kindling and attenuated the PTZ‐induced increase in NOx levels. A significant increase in BDNF levels was observed in the hippocampus of the kindled rats, which returned to the control levels following seizures induced by PTZ. 7‐NI reduced the hippocampal BDNF levels in control rats and suppressed the increase of BDNF levels in the kindled rats. Our findings suggest that NO plays a role in the development of PTZ‐induced kindling and that BDNF may contribute to the NO‐dependent plastic changes in neuronal excitability.
Central β‐amyloid peptide‐induced peripheral interleukin‐6 responses in mice
Authors: Song Dong‐Keun , Im Yeong‐Bin , Jung Jun‐Sub , Cho Jaeyoung , Suh Hong‐Won , Kim Yung‐Hi
Published: March 2003

β‐Amyloid peptides (Aβs) share with lipopolysaccharide, a potent pro‐inflammatory agent, the property of stimulating glial cells or macrophages to induce various inflammatory mediators. We recently reported that central administration of lipopolysaccharide induces peripheral interleukin‐6 responses via both the central and peripheral norepinephrine system. In this study, the effect of intracerebroventricular injection of various synthetic Aβs on plasma interleukin‐6 levels was examined in mice. Aβ1−42 dose‐dependently increased plasma interleukin‐6 levels: ‘aged’ Aβ1−42 was more effective than fresh, whereas Aβ42−1 had no effect. ‘Aged’ Aβ1−42 (205 pmol/mouse i.c.v.)‐induced plasma interleukin‐6 peaked at 2 h post injection, which is earlier than the peak time of the Aβ1−42‐induced brain interleukin‐6, tumor necrosis factor‐α and interleukin‐1β levels, which was 4, 4 and 24 h, respectively. Among various peripheral organs, Aβ1−42 (205 pmol/mouse i.c.v.) significantly increased interleukin‐6 mRNA expression in lymph nodes and liver. Aβ1−42 (205 pmol/mouse i.c.v.) significantly increased norepinephrine turnover in both hypothalamus and spleen. Either central or peripheral norepinephrine depletion effectively inhibited the Aβ1−42‐induced peripheral interleukin‐6 response. Pretreatment with prazosin (α1‐adrenergic antagonist), yohimbine (α2‐adrenergic antagonist), and ICI‐118,551 (β2‐adrenergic antagonist), but not with betaxolol (β1‐adrenergic antagonist), inhibited Aβ1−42‐induced plasma interleukin‐6 levels. These results demonstrate that centrally administered Aβ1−42 effectively induces the systemic interleukin‐6 response which is mediated, in part, by central Aβ1−42‐induced activation of the central and the peripheral norepinephrine systems.
Characterization of the in vitro phosphorylation of human tau by tau protein kinase II (cdk5/p20) using mass spectrometry
Authors: Lund Eric T. , McKenna Rosemary , Evans David B. , Sharma Satish K. , Mathews W. Rodney
Published: March 2003

Hyperphosphorylated tau is an integral part of the neurofibrillary tangles that form within neuronal cell bodies, and tau protein kinase II is reported to play a role in the pathogenesis of Alzheimer's disease. Recently, we reported that tau protein kinase II (cdk5/p20)‐phosphorylated human tau inhibits microtubule assembly, and tau protein kinase II (cdk5/p20) phosphorylation of microtubule‐associated tau results in dissociation of phosphorylated tau from the microtubules and tubulin depolymerization. In the studies reported here, a combination of mass spectrometric techniques was used to study the phosphorylation of human recombinant tau by recombinant tau protein kinase II (cdk5/p20) in vitro. The extent of phosphorylation was determined by measuring the molecular mass of phosphorylated tau using mass spectrometry. Reaction of human recombinant tau with tau protein kinase II (cdk5/p20) resulted in the formation of two major species containing either five or six phosphate groups. The specific amino acid residues phosphorylated were determined by analyzing tryptic peptides by tandem mass spectrometry via either MALDI/TOF post‐source decay or by electrospray tandem mass spectrometry. Based on these experiments, we conclude that tau protein kinase II (cdk5/p20) can phosphorylate human tau at Thr181, Thr205, Thr212, Thr217, Ser396 and Ser404.
Caspase 3 inhibition attenuates hydrogen peroxide‐induced DNA fragmentation but not cell death in neuronal PC12 cells
Authors: Jiang Dongmei , Jha Nandita , Boonplueang Rapee , Andersen Julie K.
Published: March 2003

Exposure of neurons to H2O2 results in both necrosis and apoptosis. Caspases play a pivotal role in apoptosis, but exactly how they are involved in H2O2‐mediated cell death is unknown. We examined H2O2‐induced toxicity in neuronal PC12 cells and the effects of inducible overexpression of the H2O2‐scavenging enzyme catalase on this process. H2O2 caused cell death in a time‐ and concentration‐dependent manner. Cell death induced by H2O2 was found to be mediated in part through an apoptotic pathway as H2O2‐treated cells exhibited cell shrinkage, nuclear condensation and marked DNA fragmentation. H2O2 also triggered activation of caspase 3. Genetic up‐regulation of catalase not only significantly reduced cell death but also suppressed caspase 3 activity and DNA fragmentation. While the caspase 3 inhibitor DEVD inhibited both caspase 3 activity and DNA fragmentation induced by H2O2 it did not prevent cell death. Treatment with the general caspase inhibitor ZVAD, however, resulted in complete attenuation of H2O2‐mediated cellular toxicity. These results suggest that DNA fragmentation induced by H2O2 is attributable to caspase 3 activation and that H2O2 may be critical for signaling leading to apoptosis. However, unlike inducibly increased catalase expression and general caspase inhibition both of which protect cells from cytotoxicity, caspase 3 inhibition alone did not improve cell survival suggesting that prevention of DNA fragmentation is insufficient to prevent H2O2‐mediated cell death.
Protein kinase C delta regulates neural cell adhesion molecule polysialylation state in the rat brain
Authors: Gallagher Helen C. , Murphy Keith J. , Foley Andrew G. , Regan Ciaran M.
Published: March 2003

Polysialylation of neural cell adhesion molecule (NCAM PSA) modulates cell–cell homophilic binding and signalling during brain development and the remodelling of discrete brain regions in the adult. Following learning, a transient increase in the frequency of polysialylated neurones occurs in the dentate gyrus of the hippocampal formation, and this has been correlated with the selective retention and/or elimination of synapses that are transiently overproduced during memory consolidation. We now demonstrate that protein kinase C delta (PKCδ) negatively regulates polysialyltransferase activity in the rat brain during development and also in the hippocampus during memory consolidation, where its down‐regulation in the Golgi membrane fraction coincides with the transient increase in NCAM PSA expression. Decreased expression of PKCδ was also observed in the hippocampus of rats reared in a complex environment and this directly contrasted the significant increase in frequency of hippocampal polysialylated neurones observed in these animals. These effects were isoform‐specific as no change in total PKC enzyme activity was detected during memory consolidation and complex environment rearing had no effect on the hippocampal expression of PKCα, β, γ or ε. By sequential immunoprecipitation and immunoblot analysis, phosphorylation of polysialyltransferase protein(s) was (were) demonstrated to occur on both serine and tyrosine residues and this was associated with decreased enzyme activity. Moreover, a similar experimental approach revealed the degree of PKCδ co‐precipitation with polysialyltransferase protein(s) to be inversely correlated with polysialyltransferase activity. These findings support in vitro evidence indicating PKCδ to regulate polysialyltransferase activity and NCAM polysialylation state.
γ‐Aminobutyric acidA neurotransmission and cerebral ischemia
Authors: Schwartz‐Bloom Rochelle D. , Sah Renu
Published: March 2003

In this review, we present evidence for the role of γ‐aminobutyric acid (GABA) neurotransmission in cerebral ischemia‐induced neuronal death. While glutamate neurotransmission has received widespread attention in this area of study, relatively few investigators have focused on the ischemia‐induced alterations in inhibitory neurotransmission. We present a review of the effects of cerebral ischemia on pre and postsynaptic targets within the GABAergic synapse. Both in vitro and in vivo models of ischemia have been used to measure changes in GABA synthesis, release, reuptake, GABAA receptor expression and activity. Cellular events generated by ischemia that have been shown to alter GABA neurotransmission include changes in the Cl− gradient, reduction in ATP, increase in intracellular Ca2+, generation of reactive oxygen species, and accumulation of arachidonic acid and eicosanoids. Neuroprotective strategies to increase GABA neurotransmission target both sides of the synapse as well, by preventing GABA reuptake and metabolism and increasing GABAA receptor activity with agonists and allosteric modulators. Some of these strategies are quite efficacious in animal models of cerebral ischemia, with sedation as the only unwanted side‐effect. Based on promising animal data, clinical trials with GABAergic drugs are in progress for specific types of stroke. This review attempts to provide an understanding of the mechanisms by which GABA neurotransmission is sensitive to cerebral ischemia. Furthermore, we discuss how dysfunction of GABA neurotransmission may contribute to neuronal death and how neuronal death can be prevented by GABAergic drugs.
Nerve growth factor modulates the expression and secretion of β‐amyloid precursor protein through different mechanisms in PC12 cells
Authors: Villa Ana , Latasa Maria Jesús , Pascual Angel
Published: March 2003

The β‐amyloid protein, component of the senile plaques found in Alzheimer brains is proteolytically derived from the β‐amyloid precursor protein (APP), a larger membrane‐associated protein that is expressed in both neural and non‐neural cells. Overexpression of APP might be one of the mechanisms that more directly contributes to the development of Alzheimer's disease. The APP gene expression is regulated by a number of cellular mediators including nerve growth factor (NGF) and other ligands of tyrosine kinase receptors. We have previously described that NGF increases APP mRNA levels in PC12 cells. However, the molecular mechanisms and the precise signalling pathways that mediate its regulation are not yet well understood. In the present study we present evidence that NGF, and to a lesser extent fibroblast growth factor and epidermal growth factor, stimulate APP promoter activity in PC12 cells. This induction is mediated by DNA sequences located between the nucleotides − 307 and − 15, and involves activation of the Ras–MAP kinase signalling pathway. In contrast, we have also found that NGF‐induced secretion of soluble fragments of APP into the culture medium is mediated by a Ras independent mechanism.
Leitmotifs in the biochemistry of LTP induction: amplification, integration and coordination
Authors: Dineley Kelly T. , Weeber Edwin J. , Atkins Coleen , Adams J. Paige , Anderson Anne E. , Sweatt J. David
Published: March 2003

Hippocampal long‐term potentiation (LTP) is a robust and long‐lasting form of synaptic plasticity that is the leading candidate for a cellular mechanism contributing to mammalian learning and memory. Investigations over the past decade have revealed that the biochemistry of LTP induction involves mechanisms of great subtlety and complexity. This review highlights themes that have emerged as a result of our increased knowledge of the signal transduction pathways involved in the induction of NMDA receptor‐dependent LTP in area CA1 of the hippocampus. Among these themes are signal amplification, signal integration and signal coordination. Here we use these themes as an organizing context for reviewing the profusion of signaling mechanisms involved in the induction of LTP.
Dibutyryl‐cAMP (dbcAMP) up‐regulates astrocytic chloride‐dependent l‐[3H]glutamate transport and expression of both system xc− subunits
Authors: Gochenauer Gordon E. , Robinson Michael B.
Published: March 2003

Recent studies have shown that N6,2′‐O‐dibutyryladenosine 3′:5′ cyclic monophosphate (dbcAMP) increases the expression of specific subtypes of Na+‐dependent glutamate transporters in cultured astrocytes. Our group also found that treatment of astrocytes with dbcAMP for several days increases the Na+‐independent accumulation of l‐[3H]glutamate. In this study, the properties of this Na+‐independent accumulation were characterized, and the mechanism by which dbcAMP up‐regulates this process was investigated. This accumulation was markedly reduced in the absence of Cl− and was also inhibited by several anion‐exchange inhibitors, including 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid, 4,4′‐dinitrostilbene‐2,2′‐disulfonic acid and 4‐acetamido‐4′‐isothiocyanatostilbene‐2,2′‐disulfonic acid, suggesting that this activity is mediated by a Cl−‐dependent transporter. In addition, this activity was inhibited by micromolar concentrations of several inhibitors of another Cl−‐dependent (Na+‐independent) transport activity frequently referred to as system xc− (l‐cystine, l‐α‐aminoadipate, l‐homocysteate, quisqualate, β‐N‐oxalyl‐l‐α,β‐diaminopropionate, ibotenate). This activity was competitively inhibited by several phenylglycine derivatives previously characterized as inhibitors of metabotropic glutamate receptor activation. The concentration‐dependence for Na+‐independent, Cl−‐dependent l‐[3H]glutamate uptake activity was compared for dbcAMP‐treated and untreated astrocytes. Treatment with dbcAMP increased the Vmax of this Cl−‐dependent transport activity by sixfold but had no effect on the Km value. System xc− requires two subunits, xCT and 4F2hc/CD98, to reconstitute functional activity. We found that dbcAMP caused a twofold increase in the levels of xCT mRNA and a sevenfold increase in the levels of 4F2hc/CD98 protein. This study indicates that dbcAMP up‐regulates Cl−‐dependent l‐[3H]glutamate transport activity in astrocytes and suggests that this effect is related to increased expression of both subunits of system xc−. Because this activity is thought to be important for the synthesis of glutathione and protection from oxidant injury, understanding the regulation of system xc− may provide alternate approaches to limit this form of injury.
Retracted: Neuroprotection mediated by glial group‐II metabotropic glutamate receptors requires the activation of the MAP kinase and the phosphatidylinositol‐3‐kinase pathways
Authors: D'Onofrio M. , Cuomo L. , Battaglia G. , Ngomba R. T. , Storto M. , Kingston A. E. , Orzi F. , De Blasi A. , Di Iorio P. , Nicoletti F. , Bruno V.
Published: March 2003

The mGlu2/3 receptor agonists 4‐carboxy‐3‐hydroxyphenylglycine (4C3HPG) and LY379268 attenuated NMDA toxicity in primary cultures containing both neurons and astrocytes. Neuroprotection was abrogated by PD98059 and LY294002, which inhibit the mitogen activated protein kinase (MAPK) and the phosphatidylinositol‐3‐kinase (PI‐3‐K) pathways, respectively. Cultured astrocytes lost the ability to produce transforming growth factor‐β1 (TGF‐β1) in response to mGlu2/3 receptor agonists when co‐incubated with PD98059 or LY294002. As a result, the glial medium was no longer protective against NMDA toxicity. Activation of the MAPK and PI‐3‐K pathways in cultured astrocytes treated with 4C3HPG or LY379268 was directly demonstrated by an increase in the phosphorylated forms of ERK‐1/2 and Akt. Similarly to that observed in the culture, intracerebral or systemic injections of mGlu2/3 receptor agonists enhanced TGF‐β1 formation in the rat or mouse caudate nucleus, and this effect was reduced by PD98059. PD98059 also reduced the ability of LY379268 to protect striatal neurons against NMDA toxicity. These results suggest that activation of glial mGlu2/3 receptors induces neuroprotection through the activation of the MAPK and PI‐3‐K pathways leading to the induction of TGF‐β.
Proteasomal inhibition leads to formation of ubiquitin/α‐synuclein‐immunoreactive inclusions in PC12 cells
Authors: Rideout Hardy J. , Larsen Kristin E. , Sulzer David , Stefanis Leonidas
Published: March 2003

Proteasomal dysfunction has been recently implicated in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease and diffuse Lewy body disease. We have developed an in vitro model of proteasomal dysfunction by applying pharmacological inhibitors of the proteasome, lactacystin or ZIE[O‐tBu]‐A‐leucinal (PSI), to dopaminergic PC12 cells. Proteasomal inhibition caused a dose‐dependent increase in death of both naive and neuronally differentiated PC12 cells, which could be prevented by caspase inhibition or CPT‐cAMP. A percentage of the surviving cells contained discrete cytoplasmic ubiquitinated inclusions, some of which also contained synuclein‐1, the rat homologue of human α‐synuclein. However the total level of synuclein‐1 was not altered by proteasomal inhibition. The ubiquitinated inclusions were present only within surviving cells, and their number was increased if cell death was prevented. We have thus replicated, in this model system, the two cardinal pathological features of Lewy body diseases, neuronal death and the formation of cytoplasmic ubiquitinated inclusions. Our findings suggest that inclusion body formation and cell death may be dissociated from one another.
Differences in survival‐promoting effects and intracellular signaling properties of BDNF and IGF‐1 in cultured cerebral cortical neurons
Authors: Yamada Masashi , Tanabe Keiko , Wada Kazuyo , Shimoke Koji , Ishikawa Yasuyuki , Ikeuchi Toshihiko , Koizumi Shinichi , Hatanaka Hiroshi
Published: March 2003

Brain‐derived neurotrophic factor (BDNF) and insulin‐like growth factor‐1 (IGF‐1) act on various neurons of the CNS as neurotrophic factors promoting neuronal differentiation and survival. We examined the survival‐promoting effects of BDNF and IGF‐1 on serum deprivation‐induced death in cultured cerebral cortical neurons, and compared the intracellular signaling pathways stimulated by BDNF and IGF‐1 in the neurons. We found that the survival‐promoting effect of BDNF was much weaker than that of IGF‐1 in serum deprivation‐induced death of cultured cortical neurons. We found no differences in the levels of phosphatidylinositol 3‐kinase (PtdIns3‐K) activity or Akt (also called PKB) phosphorylation induced by BDNF and IGF‐1 in the cultured cortical neurons, although many reports suggest that PtdIns3‐K and Akt are involved in survival promotion. In addition, phosphorylation signals of mitogen‐activated protein kinase (MAPK) and cAMP responsive element‐binding protein (CREB), which have also been reported to be involved in survival promotion, were stimulated by BDNF much more potently than by IGF‐1. These results show that there may be, as yet unidentified, intracellular signaling pathways other than the PtdIns3‐K‐Akt, MAPK and CREB signaling, to regulate survival promotion. These unidentified signaling pathways may be responsible for the distinct strengths of the survival‐promoting effects of BDNF and IGF‐1.
Catecholamine release and uptake in the mouse prefrontal cortex
Authors: Mundorf Michelle L. , Joseph Joshua D. , Austin C. Megan , Caron Marc G. , Wightman R. Mark
Published: March 2003

Monitoring the release and uptake of catecholamines from terminals in weakly innervated brain regions is an important step in understanding their importance in normal brain function. To that end, we have labeled brain slices from transgenic mice that synthesize placental alkaline phosphatase (PLAP) on neurons containing tyrosine hydroxylase with antibody–fluorochrome conjugate, PLAP‐Cy5. Excitation of the fluorochrome enables catecholamine neurons to be visualized in living tissue. Immunohistochemical fluorescence with antibodies to tyrosine hydroxylase and dopamine β‐hydroxylase revealed that the PLAP labeling was specific to catecholamine neurons. In the prefrontal cortex (PFC), immunohistochemical fluorescence of the PLAP along with staining for dopamine transporter (DAT) and norepinephrine transporter (NET) revealed that all three exhibit remarkable spatial overlap. Fluorescence from the PLAP antibody was used to position carbon‐fiber microelectrodes adjacent to catecholamine neurons in the PFC. Following incubation with l‐DOPA, catecholamine release and subsequent uptake was measured and the effect of uptake inhibitors examined. Release and uptake in NET and DAT knockout mice were also monitored. Uptake rates in the cingulate and prelimbic cortex are so slow that catecholamines can exist in the extracellular fluid for sufficient time to travel ∼100 µm. The results support heterologous uptake of catecholamines and volume transmission in the PFC of mice.
Cannabinoid receptor agonists inhibit depolarization‐induced calcium influx in cerebellar granule neurons
Authors: Nogueron M. Isabel , Porgilsson Bjorn , Schneider Wendy E. , Stucky Cheryl L. , Hillard Cecilia J.
Published: March 2003

Neuronal cannabinoid receptors (CB1) are coupled to inhibition of voltage‐sensitive Ca2+ channels (VSCCs) in several cell types. The purpose of these studies was to characterize the interaction between endogenous CB1 receptors and VSCCs in cerebellar granule neurons (CGN). Ca2+ transients were evoked by KCl‐induced depolarization and imaged using fura‐2. The CB1 receptor agonists CP55940, Win 55212–2 and N‐arachidonylethanolamine (anandamide) produced concentration‐related decreases in peak amplitude of the Ca2+ response and total Ca2+ influx. Pre‐treatment of CGN with pertussis toxin abolished agonist‐mediated inhibition. The inhibitory effect of Win 55212–2 on Ca2+ influx was additive with inhibition produced by ω‐agatoxin IVA and nifedipine but not with ω‐conotoxin GVIA, indicating that N‐type VSCCs are the primary effector. Paradoxically, the CB1 receptor antagonist, SR141716, also inhibited KCl‐induced Ca2+ influx into CGN in a concentration‐related manner. SR141716 inhibition was pertussis toxin‐insensitive and was not additive with the inhibition produced by Win 55212–2. Confocal imaging of CGN in primary culture demonstrate a high density of CB1 receptor expression on CGN plasma membranes, including the neuritic processes. These data demonstrate that the CB1 receptor is highly expressed by CGN and agonists serve as potent and efficacious inhibitory modulators of Ca2+ influx through N‐type VSCC.
Nigrostriatal denervation does not affect glutamate transporter mRNA expression but subsequent levodopa treatment selectively increases GLT1 mRNA and protein expression in the rat striatum
Authors: Liévens J.‐C. , Salin P. , Nieoullon A. , Kerkerian‐Le Goff L.
Published: March 2003

There is growing evidence that the loss of the nigrostriatal dopaminergic neurones induces an overactivity of the corticostriatal glutamatergic pathway which seems to be central to the physiopathology of parkinsonism. Moreover, glutamatergic mechanisms involving NMDA receptors have been shown to interfere with the therapeutical action of levodopa. Given the key role played by uptake processes in glutamate neurotransmission, this study examined the effects of nigrostriatal deafferentation and of levodopa treatment on the striatal expression of the glutamate transporters GLT1, GLAST and EAAC1 in the rat. No significant changes in striatal mRNA levels of these transporters were detected after either levodopa treatment (100 mg/kg; i.p., twice a day for 21 days) or unilateral lesion of the nigrostriatal pathway by intranigral 6‐hydroxydopamine injection. In contrast, animals with the lesion subsequently treated with levodopa showed a selective increase (36%) in GLT1 mRNA levels in the denervated striatum versus controls. These animals also showed increased GLT1 protein expression, as assessed by immunostaining and western blotting. These data provide the first evidence that levodopa therapy may interfere with striatal glutamate transmission through change in expression of the primarily glial glutamate transporter GLT1. We further suggest that levodopa‐induced GLT1 overexpression may represent a compensatory mechanism preventing neurotoxic accumulation of endogenous glutamate.
Oxidative stress in Ca2+‐induced membrane permeability transition in brain mitochondria
Authors: Maciel Evelise N. , Vercesi Anibal E. , Castilho Roger F.
Published: March 2003

Mitochondrial permeability transition (PT) is a non‐selective inner membrane permeabilization, typically promoted by the accumulation of excessive quantities of Ca2+ ions in the mitochondrial matrix. This phenomenon may contribute to neuronal cell death under some circumstances, such as following brain trauma and hypoglycemia. In this report, we show that Ca2+‐induced brain mitochondrial PT was stimulated by Na+ (10 mm) and totally prevented by the combination of ADP and cyclosporin A. Removal of Ca2+ from the mitochondrial suspension by EGTA or inhibition of Ca2+ uptake by ruthenium red partially reverted the dissipation of the membrane potential associated with PT. Ca2+‐induced brain mitochondrial PT was significantly inhibited by the antioxidant catalase, indicating the participation of reactive oxygen species in this process. An increased detection of reactive oxygen species, measured through dichlorodihydrofluorescein oxidation, was observed after mitochondrial Ca2+ uptake. Ca2+‐induced dichlorodihydrofluorescein oxidation was enhanced by Na+ and prevented by ADP and cyclosporin A, indicating that PT enhances mitochondrial oxidative stress. This could be at least in part a consequence of the extensive depletion in NAD(P)H that accompanied this Ca2+‐induced mitochondrial PT. NADPH is known to maintain the antioxidant function of the glutathione reductase/peroxidase and thioredoxin reductase/peroxidase systems. In addition, the occurrence of mitochondrial PT was associated with membrane lipid peroxidation. We conclude that PT further increases Ca2+‐induced oxidative stress in brain mitochondria leading to secondary damage such as lipid peroxidation.
Norepinephrine triggers Ca2+‐dependent exocytosis of 5‐hydroxytryptamine from rat pinealocytes in culture
Authors: Yamada Hiroshi , Hayashi Mitsuko , Uehara Shunsuke , Kinoshita Mika , Muroyama Akiko , Watanabe Masami , Takei Koji , Moriyama Yoshinori
Published: March 2003

5‐Hydroxytryptamine (5‐HT) is a precursor and a putative modulator for melatonin synthesis in mammalian pinealocytes. 5‐HT is present in organelles distinct from l‐glutamate‐containing synaptic‐like microvesicles as well as in the cytoplasm of pinealocytes, and is secreted upon stimulation by norepinephrine (NE) to enhance serotonin N‐acetyltransferase activity via the 5‐HT2 receptor. However, the mechanism underlying the secretion of 5‐HT from pinealocytes is unknown. In this study, we show that NE‐evoked release of 5‐HT is largely dependent on Ca2+ in rat pinealocytes in culture. Omission of Ca2+ from the medium and incubation of pineal cells with EGTA‐tetraacetoxymethyl‐ester inhibited by 59 and 97% the NE‐evoked 5‐HT release, respectively. Phenylephrine also triggered the Ca2+‐dependent release of 5‐HT, which was blocked by phentolamine, an α antagonist, but not by propranolol, a β antagonist. Botulinum neurotoxin type E cleaved 25 kDa synaptosomal‐associated protein and inhibited by 50% of the NE‐evoked 5‐HT release. Bafilomycin A1, an inhibitor of vacuolar H+‐ATPase, and reserpine and tetrabenazine, inhibitors of vesicular monoamine transporter, all decreased the storage of vesicular 5‐HT followed by inhibition of the NE‐evoked 5‐HT release. Agents that trigger l‐glutamte exocytosis such as acetylcholine did not trigger any Ca2+‐dependent 5‐HT release. Vice versa neither NE nor phenylephrine caused synaptic‐like microvesicle‐mediated l‐glutamate exocytosis. These results indicated that upon stimulation of a adrenoceptors pinealocytes secrete 5‐HT through a Ca2+‐dependent exocytotic mechanism, which is distinct from the exocytosis of synaptic‐like microvesicles.
Expression of Cdk5 and its activators in NT2 cells during neuronal differentiation
Authors: Fu Wing‐Yu , Wang Jerry H. , Ip Nancy Y.
Published: March 2003

We have recently developed a rapid protocol involving NT2 cell aggregation and treatment with retinoic acid (RA) to produce terminally differentiated CNS neurons. As a first step to explore the functional roles of cell‐cycle regulatory proteins in the process of neuronal differentiation, the expression profiles of cyclin‐dependent kinases (Cdks) and their regulators were examined in NT2 cells following treatment with RA. One of the Cdks, Cdk5, has been demonstrated to affect the process of neuronal differentiation and suggested to play an important role in development of the nervous system. We found that the expression of Cdk5 was gradually increased, while its activators (p35 and p39) as well as Cdk5 kinase activity were induced in NT2 cells during the process of neuronal differentiation. Moreover, both p35 and p39 were localized along the axons and varicosity‐like structures of differentiated NT2 neurons. Taken together, our results demonstrated that NT2 cells provide a good in vitro model system to examine signaling pathways involved in the regulation of Cdk5 activators and to elucidate the functional roles of Cdk5 in neuronal differentiation.
Profound molecular changes following hippocampal slice preparation: loss of AMPA receptor subunits and uncoupled mRNA/protein expression
Authors: Taubenfeld Stephen M. , Stevens Kimberly A. , Pollonini Gabriella , Ruggiero Jason , Alberini Cristina M.
Published: March 2003

The acute hippocampal slice preparation is a convenient, in vitro model widely used to study the biological basis of synaptic plasticity. Although slices may preserve their electrophysiological properties for several hours, profound molecular changes in response to the injury caused by the slicing procedure are likely to occur. To determine the magnitude and duration of these changes we examined the post‐slicing expression kinetics of three classes of genes known to be implicated in long‐term synaptic plasticity: glutamate AMPA receptors (GluR), transcription factors and neurotrophins. Slicing resulted in a striking loss of GluR1 and GluR3, but not of GluR2 proteins suggesting that rapid changes in the composition of major neurotransmitter receptors may occur. Slicing caused a significant induction of the transcription factors c‐fos, zif268, CCAAT enhancer binding protein (C/EBP) β and δ mRNAs and of the neurotrophin brain‐derived neurothophic factor (BDNF) mRNA. In contrast, there was no augmentation, and sometimes a decline, in the levels of the corresponding proteins. These data reveal that significant discrepancies exist between the slice preparation and the intact hippocampus in terms of the metabolism of molecular components known to be involved in synaptic plasticity.
Intracellular distribution of the fluorescent dye nonyl acridine orange responds to the mitochondrial membrane potential: implications for assays of cardiolipin and mitochondrial mass
Authors: Jacobson Jake , Duchen Michael R. , Heales Simon J. R.
Published: March 2003

Cardiolipin, a polyunsaturated acidic phospholipid, is found exclusively in bacterial and mitochondrial membranes where it is intimately associated with the enzyme complexes of the respiratory chain. Cardiolipin structure and concentration are central to the function of these enzyme complexes and damage to the phospholipid may have consequences for mitochondrial function. The fluorescent dye, 10 nonyl acridine orange (NAO), has been shown to bind cardiolipin in vitro and is frequently used as a stain in living cells to assay cardiolipin content. Additionally, NAO staining has been used to measure the mitochondrial content of cells as dye binding to mitochondria is reportedly independent of the membrane potential. We used confocal microscopy to examine the properties of NAO in cortical astrocytes, neonatal cardiomyocytes and in isolated brain mitochondria. We show that NAO, a lipophilic cation, stained mitochondria selectively. However, the accumulation of the dye was clearly dependent upon the mitochondrial membrane potential and depolarisation of mitochondria induced a redistribution of dye. Moreover, depolarisation of mitochondria prior to NAO staining also resulted in a reduced NAO signal. These observations demonstrate that loading and retention of NAO is dependant upon membrane potential, and that the dye cannot be used as an assay of either cardiolipin or mitochondrial mass in living cells.
AMPA receptor‐mediated toxicity in oligodendrocyte progenitors involves free radical generation and activation of JNK, calpain and caspase 3
Authors: Liu Hsueh‐Ning , Giasson Benoit I. , Mushynski Walter E. , Almazan Guillermina
Published: March 2003

The molecular mechanisms underlying AMPA (α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate) receptor‐mediated excitotoxicity were characterized in rat oligodendrocyte progenitor cultures. Activation of AMPA receptors, in the presence of cyclothiazide to selectively block desensitization, produced a massive Ca2+ influx and cytotoxicity which were blocked by the antagonists CNQX and GYKI 52466. A role for free radical generation in oligodendrocyte progenitor cell death was deduced from three observations: (i) treatment with AMPA agonists decreased intracellular glutathione; (ii) depletion of intracellular glutathione with buthionine sulfoximine potentiated cell death; and (iii) the antioxidant N‐acetylcysteine replenished intracellular glutathione and protected cultures from AMPA receptor‐mediated toxicity. Cell death displayed some characteristics of apoptosis, including DNA fragmentation, chromatin condensation and activation of caspase‐3 and c‐Jun N‐terminal kinase (JNK). A substrate of calpain and caspase‐3, α‐spectrin, was cleaved into characteristic products following treatment with AMPA agonists. In contrast, inhibition of either caspase‐3 by DEVD‐CHO or calpain by PD 150606 protected cells from excitotoxicity. Our results indicate that overactivation of AMPA receptors causes apoptosis in oligodendrocyte progenitors through mechanisms involving Ca2+ influx, depletion of glutathione, and activation of JNK, calpain, and caspase‐3.