bluuebunny/crossref_metadata_2025 huggingface dataset:
The HIV‐1 Nef Protein Inhibits Extracellular Signal‐Regulated Kinase‐Dependent DNA Synthesis in a Human Astrocytic Cell Line
Authors: Romero Ignacio A. , Teixeira Alexandra , Strosberg A. Donny , Cazaubon Sylvie , Couraud Pierre‐Olivier
Published: July 2010

Abstract: The role of nonproductive infection of astrocytes by human immunodeficiency virus type 1 (HIV‐1), characterized by the overexpression of nef, in brain disease progression is largely unknown. We investigated the consequences of stable expression of nef from the HIV‐1 strain LAI in the human astrocytic cell line U373. DNA synthesis induced by endothelin‐1 (ET‐1) was largely decreased by nef. Stable expression of nef did not affect the ET‐1‐induced tyrosine phosphorylation of focal adhesion kinase, an adhesion‐dependent pathway known to participate in DNA synthesis in astrocytes. Conversely, the activation of extracellular signal‐regulated kinase (ERK) by ET‐1 was largely inhibited in cells stably or transiently expressing nef. A similar inhibitory action of nef on ERK activation was observed after direct stimulation of G proteins. Furthermore, the inhibitory action of nef did not require protein kinase C (PKC) and affected mainly the PKC‐independent pathway of ERK activation. Following chemokine receptor CXCR4‐mediated infection of U373 cells stably expressing CXCR4 with the T‐tropic HIV‐1 strain m7‐NDK, ET‐1‐induced activation of ERK was also inhibited. Altogether, these results indicate that intracellular signaling pathways associated with the growth factor activity of ET‐1 are impaired in nef‐expressing and HIV‐1‐infected astrocytes, suggesting that infection of astrocytes may play a significant role in the neuropathogenesis of HIV‐1 encephalopathy.
Expression of GABA Receptor ρ Subunits in Rat Brain
Authors: Boue‐Grabot Eric , Roudbaraki Morad , Bascles Lionel , Tramu Gerard , Bloch Bertrand , Garret Maurice
Published: July 2010

Abstract: The GABA receptor ρ1, ρ2, and ρ3 subunits are expressed in the retina where they form bicuculline‐insensitive GABAC receptors. We used northern blot, in situ hybridization, and RT‐PCR analysis to study the expression of ρ subunits in rat brains. In situ hybridization allowed us to detect ρ‐subunit expression in the superficial gray layer of the superior colliculus and in the cerebellar Purkinje cells. RT‐PCR experiments indicated that (a) in retina and in domains that may contain functional GABAC receptors, ρ2 and ρ1 subunits are expressed at similar levels; and (b) in domains and in tissues that are unlikely to contain GABAC receptors, ρ2 mRNA is enriched relative to ρ1 mRNA. These results suggest that both ρ1 and ρ2 subunits are necessary to form a functional GABAC receptor. The use of RT‐PCR also showed that, except in the superior colliculus, ρ3 is expressed along with ρ1 and ρ2 subunits. We also raised an antibody against a peptide sequence unique to the ρ1 subunit. The use of this antibody on cerebellum revealed the rat ρ1 subunit in the soma and dendrites of Purkinje neurons. The allocation of GABAC receptor subunits to identified neurons paves the way for future electrophysiological studies.
Stimulus‐Specific Hierarchy of Enhancer Elements Within the Rat Prodynorphin Promoter
Authors: Carrión Angel M. , Mellström Britt , Luckman Simon M. , Naranjo Jose R.
Published: July 2010

Abstract: Induction of the prodynorphin gene occurs in a tissue‐specific manner following different physiological stimuli. Using electrophoretic mobility shift assays, we studied the relative activity of the five major regulatory sites in the rat prodynorphin promoter. Prodynorphin cyclic AMP‐responsive element 2 (DynCRE2), DynCRE3, and the noncanonical prodynorphin AP‐1 (ncDynAP‐1) regulatory sites control, in a coordinated manner, prodynorphin induction in the spinal cord after noxious stimulation, whereas prodynorphin up‐regulation in supraoptic neurons is regulated predominantly by the ncDynAP‐1. Conversely, prodynorphin transactivation in the ovaries upon gonadotrophin stimulation is controlled by DynCRE1 and DynCRE3. Our results support the idea that stimulus‐specific changes in nuclear proteins establish a functional hierarchy among regulatory sites in the prodynorphin promoter and provide further insight in the molecular mechanisms that govern prodynorphin gene regulation.
Functional Characterization of the Murine Serotonin Transporter Gene Promoter in Serotonergic Raphe Neurons
Authors: Heils Armin , Wichems Christine , Mössner Rainald , Petri Susanne , Glatz Katharina , Bengel Dietmar , Murphy Dennis L. , Lesch Klaus‐Peter
Published: July 2010

Abstract: We have isolated and characterized the 5′‐flanking regulatory region of the murine serotonin 5‐HT transporter (5‐HTT) gene. A TATA‐like motif and several potential binding sites for transcription factors, including two AP1, several AP2 and AP4 binding sites, CCAAT and GC boxes (SP1 binding sites), a nuclear factor‐κB, and a cyclic AMP response element‐like motif, are present in the 5′‐flanking region. A ∼2.2‐kb fragment (−2,143 to +51 with respect to the transcription start site), which had been fused to the luciferase reporter gene and transiently expressed in a 5‐HTT‐expressing cell line and in serotonergic raphe neurons derived from embryonic rat brain‐stem, displayed both constitutive and inducible promoter activity. Functional promoter mapping revealed two clusters of activating elements from bp −82 to −527 and bp −1,001 to −1,937. A cell/neuron‐selective silencer element(s) is contained between bp −294 and −527. Our findings suggest that (1) the murine 5‐HTT gene promoter is active in serotonergic raphe neurons but significantly repressed in neuronal cells from frontal cortex that do not express 5‐HTT, (2) the information contained within ∼0.5 kb of the 5′‐flanking sequence is sufficient to confer its cell‐selective expression, (3) the promoter responds to cyclic AMP‐ and protein kinase C‐dependent induction, and (4) the expression of the 5‐HTT is regulated by a combination of positive and negative cis‐acting elements operating through a basal promoter unit defined by a TATA‐like motif. Fusion of the 5‐HTT gene promoter unit to a gene of choice may aid its cell‐selective expression in transgenic strategies.
Cytoskeletal and Phosphoinositide Requirements for Muscarinic Receptor Signaling to Focal Adhesion Kinase and Paxillin
Authors: Linseman Daniel A. , McEwen Edward L. , Sorensen Scott D. , Fisher Stephen K.
Published: July 2010

Abstract: The mechanism whereby agonist occupancy of muscarinic cholinergic receptors elicits an increased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin has been examined. Addition of oxotremorine‐M to SH‐SY5Y neuroblastoma cells resulted in rapid increases in the phosphorylation of FAK (t1/2 = 2 min) and paxillin that were independent of integrin‐extracellular matrix interactions, cell attachment, and the production of phosphoinositide‐derived second messengers. In contrast, the increased tyrosine phosphorylations of FAK and paxillin were inhibited by inclusion of either cytochalasin D or mevastatin, agents that disrupt the cytoskeleton. Furthermore, phosphorylation of FAK and paxillin could be prevented by addition of either wortmannin or LY‐294002, under conditions in which the synthesis of phosphatidylinositol 4‐phosphate was markedly attenuated. These results indicate that muscarinic receptor‐mediated increases in the tyrosine phosphorylation of FAK and paxillin in SH‐SY5Y neuroblastoma cells depend on both the maintenance of an actin cytoskeleton and the ability of these cells to synthesize phosphoinositides.
Astrocytic Gap Junctional Communication Decreases Neuronal Vulnerability to Oxidative Stress‐Induced Disruption of Ca2+ Homeostasis and Cell Death
Authors: Blanc Emmanuelle M. , Bruce‐Keller Annadora J. , Mattson Mark P.
Published: July 2010

Abstract: We investigated the effect of uncoupling astrocytic gap junctions on neuronal vulnerability to oxidative injury in embryonic rat hippocampal cell cultures. Mixed cultures (neurons growing on an astrocyte monolayer) treated with 18‐α‐glycyrrhetinic acid (GA), an uncoupler of gap junctions, showed markedly enhanced generation of intracellular peroxides (2,7‐dichlorofluorescein fluorescence), impairment of mitochondrial function [(dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide reduction], and cell death (lactate dehydrogenase release) following exposure to oxidative insults (FeSO4 and 4‐hydroxynonenal). GA alone had little or no effect on basal levels of peroxides, mitochondrial function, or neuronal survival. Intercellular dye transfer analyses revealed extensive astrocyte‐astrocyte coupling but no astrocyte‐neuron or neuron‐neuron coupling in the mixed cultures. Studies of pure astrocyte cultures and microscope analyses of neurons in mixed cultures showed that the increased oxidative stress and cell death in GA‐treated cultures occurred only in neurons and not in astrocytes. Antioxidants (propyl gallate and glutathione) blocked the death of neurons exposed to FeSO4/GA. Elevations of neuronal intracellular calcium levels ([Ca2+]i) induced by FeSO4 were enhanced in neurons in mixed cultures exposed to GA. Removal of extracellular Ca2+ and the L‐type Ca2+ channel blocker nimodipine prevented impairment of mitochondrial function and cell death induced by FeSO4 and GA, whereas glutamate receptor antagonists were ineffective. Finally, GA exacerbated kainate‐ and FeSO4‐induced injury to pyramidal neurons in organotypic hippocampal slice cultures. The data suggest that interastrocytic gap junctional communication decreases neuronal vulnerability to oxidative injury by a mechanism involving stabilization of cellular calcium homeostasis and dissipation of oxidative stress.
Selective Increase of α2A‐Adrenoceptor Agonist Binding Sites in Brains of Depressed Suicide Victims
Authors: Callado Luis F. , Meana J. Javier , Grijalba Bernardo , Pazos Angel , Sastre Magdalena , García‐Sevilla Jesús A.
Published: July 2010

Abstract: The α2A‐ and α2C‐adrenoceptor subtypes were evaluated in postmortem brains from suicides with depression (n = 22), suicides with other diagnoses (n = 12), and controls (n = 26). Membrane assays with the antagonist [3H]RX821002 (2‐[3H]methoxyidazoxan) suggested the presence of α2A‐adrenoceptors in the frontal cortex and both α2C‐adrenoceptors and α2A‐adrenoceptors in the caudate. The proportions in caudate were similar in controls (α2A, 86%; α2C, 14%), depressed suicides (α2A, 91%; α2C, 9%), and suicides with other diagnoses (α2A, 88%; α2C, 12%). Autoradiography of [3H]RX821002 binding under α2B/C‐adrenoceptor‐masking conditions confirmed the similar densities of α2A‐adrenoceptors in the cortex, hippocampus, and striatum from controls and suicides. In the frontal cortex of depressed suicides, competition of [3H]RX821002 binding by (−)‐adrenaline revealed a greater proportion (61 ± 9%) of α2A‐adrenoceptors in the high‐affinity conformation for agonists than in controls (39 ± 5%). Simultaneous analysis with the agonists [3H]clonidine and [3H]UK14304 and the antagonist [3H]RX821002 in the same depressed suicides confirmed the enhanced α2A‐adrenoceptor density when evaluated by agonist, but not by antagonist, radioligands. The results indicate that depression is associated with a selective increase in the high‐affinity conformation of the brain α2A‐adrenoceptors.
Okadaic Acid‐Induced Apoptosis in Neuronal Cells: Evidence for an Abortive Mitotic Attempt
Authors: Nuydens Rony , De Jong Mirjam , Van Den Kieboom Gerd , Heers Cara , Dispersyn Gwenda , Cornelissen Frans , Nuyens Roger , Borgers Marcel , Geerts Hugo
Published: July 2010

Abstract: There is increasing evidence that apoptosis in postmitotic neurons is associated with a frustrated attempt to reenter the mitotic cycle. Okadaic acid, a specific protein phosphatase inhibitor, is currently used in models of Alzheimer's research to increase the degree of phosphorylation of various proteins, such as the microtubule‐associated protein tau. Okadaic acid induces programmed cell death in the human neuroblastoma cell lines TR14 and NT2‐N, as evidenced by fragmentation of DNA and attenuation of this process by protein synthesis inhibitors. In differentiated TR14 cells, okadaic acid increases the fraction of cells in the S phase, induces the appearance of cyclin B1 and cyclin D1 markers of the cell cycle, and triggers a time‐dependent increase in DNA fragmentation after release of a thymidine block. Fully differentiated NT2‐N cells are forced to enter the mitotic cycle as shown by DNA staining. Chromatin condensation and chromosome formation are initiated, but the cells fail to complete their mitotic cycle. These data suggest that okadaic acid forces differentiated neuronal cells into the mitotic cycle. This pattern of cyclin up‐regulation and cell cycle shift is compared with apoptosis induced by neurotrophic factor deprivation in differentiated rat pheochromocytoma PC12 cells.
Immunochemical Characterization of the Deficiency of the α‐Ketoglutarate Dehydrogenase Complex in Thiamine‐Deficient Rat Brain
Authors: Sheu Kwan‐Fu Rex , Calingasan Noel Y. , Lindsay J. Gordon , Gibson Gary E.
Published: July 2010

Abstract: Mitochondrial dysfunction is a common feature of many neurodegenerative disorders. The metabolic encephalopathy caused by thiamine deficiency (TD) is a classic example in which an impairment of cerebral oxidative metabolism leads to selective cell death. In experimental TD in rodents, a reduction in the activity of the thiamine diphosphate‐dependent, mitochondrial enzyme α‐ketoglutarate dehydrogenase complex (KGDHC) occurs before the onset of pathologic lesions and is among the earliest biochemical deficits found. To understand the molecular basis and the significance of the deficiency of KGDHC in TD‐induced brain damage, the enzyme activity and protein levels of KGDHC were analyzed. The effect of TD on the subregional/cellular distribution of KGDHC and the anatomic relation of KGDHC with selective cell death were also tested by immunocytochemistry. Consistent with several previous studies, TD dramatically reduced KGDHC activity in both anatomically damaged (thalamus and inferior colliculus) and spared (cerebral cortex) regions. Immunocytochemistry revealed no apparent correlation of regional KGDHC immunoreactivity or its response to TD with affected regions in TD. The basis of the enzymatic and immunocytochemical behavior of KGDHC was further assessed by quantitative immunoblots, using antibodies specific for each of the three KGDHC components. Despite the marked decrease of KGDHC activity in TD, no reduction of any of the three KGDHC protein levels was found. Thus, TD impairs the efficacy of the KGDHC catalytic machinery, whereas the concentration of protein molecules persists. The generalized decline of KGDHC activity with no apparent anatomic selectivity is consistent with the notion that the compromised mitochondrial oxidation sensitizes the brain cells to various other insults that precipitate the cell death. The current TD model provides a relevant experimental system to understand the molecular basis of many neurodegenerative conditions in which mitochondrial dysfunction and KGDHC deficiency are prominent features.
Effect of Aluminum on Acetyl‐CoA and Acetylcholine Metabolism in Nerve Terminals
Authors: Bielarczyk Hanna , Tomaszewicz Maria , Szutowicz Andrzej
Published: July 2010

Abstract: The potential ability of Al to affect cholinergic transmission was studied on synaptosomal fractions of rat brain incubated with pyruvate in depolarizing medium containing 30 mM K+. Addition of 1 mM Ca caused a 266% increase in the acetylcholine (ACh) release despite decreased pyruvate oxidation. Under these conditions, 0.25 mM Al did not affect pyruvate oxidation but raised mitochondrial and decreased synaptoplasmic acetyl‐CoA. Simultaneously, a 61% inhibition of Ca‐evoked ACh release was observed. Verapamil (0.1 and 0.5 mM) decreased the acetyl‐CoA concentration in synaptoplasm and inhibited ACh release. Al (0.012 mM) partially reversed these inhibitory effects. Omission of Pi from the medium abolished suppressive effects of Al on acetyl‐CoA content and Ca‐evoked transmitter release. We conclude that the Al(PO4)OH− complex may be the active form of Al, which, by interaction with the verapamil binding sites of Ca channels, is likely to restrict the Ca influx to the synaptoplasm. This may inhibit the provision of acetyl‐CoA to the synaptoplasm as well as the Ca‐evoked ACh release. One may suppose that excessive accumulation of Al in some encephalopathic brains may, by this mechanism, suppress still‐surviving cholinergic neurons and exacerbate cognitive deficits caused by already‐existing structural losses in the cholinergic system.
Depolarization Differentially Affects Allosteric Modulation by Neurotoxins of Scorpion α‐Toxin Binding on Voltage‐Gated Sodium Channels
Authors: Cestèle Sandrine , Gordon Dalia
Published: July 2010

Abstract: Voltage‐gated sodium channels serve as a target for many neurotoxins that bind to several distinct, allosterically interacting receptor sites. We examined the effect of membrane potentials (incited by increasing external K+ concentrations) on the binding modulation by veratridine, brevetoxin, and tetrodotoxin of the scorpion α‐toxin AaH II to receptor site 3 on sodium channels of rat brain synaptosomes. Depolarization is shown to differentially modulate neurotoxin effects on AaH II binding: Veratridine increase is potentiated, brevetoxin's inhibitory effect is reduced, and tetrodotoxin enhancement is evident mainly at resting membrane potential (5 mM K+). Both tetrodotoxin and veratridine apparently reverse the inhibition of AaH II binding by brevetoxin at resting membrane potential, but only veratridine is able to partially restore AaH II binding at 0 mV (135 mM K+). Thus, the allosteric interactions are grouped into two categories, depending on the membrane potential. Under depolarized conditions, the cooperative effects among veratridine and brevetoxin on AaH II binding fit the previously described two‐state conformational model. At resting membrane potential, additional interactions are revealed, which may be explained by assuming that toxin binding induces conformational changes on the channel structure, in addition to being state‐dependent. Our results provide a new insight into neurotoxin action and the complex dynamic changes underlying allosteric coupling of neurotoxin receptor sites, which may be related to channel gating.
Novel Large Apolipoprotein E‐Containing Lipoproteins of Density 1.006–1.060 g/ml in Human Cerebrospinal Fluid
Authors: Guyton John R. , Miller Sara E. , Martin Margaret E. , Khan Wasiuddin A. , Roses Allen D. , Strittmatter Warren J.
Published: July 2010

Abstract: Although the critical role of apolipoprotein E (apoE) allelic variation in Alzheimer's disease and in the outcome of CNS injury is now recognized, the functions of apoE in the CNS remain obscure, particularly with regard to lipid metabolism. We used density gradient ultracentrifugation to identify apoE‐containing lipoproteins in human CSF. CSF apoE lipoproteins, previously identified only in the 1.063–1.21 g/ml density range, were also demonstrated in the 1.006–1.060 g/ml density range. Plasma lipoproteins in this density range include low‐density lipoprotein and high‐density lipoprotein (HDL) subfraction 1 (HDL1). The novel CSF apoE lipoproteins are designated HDL1. No immunoreactive apolipoprotein A‐I (apo A‐I) or B could be identified in the CSF HDL1 fractions. Large lipoproteins 18.3 ± 6.6 nm in diameter (mean ± SD) in the HDL1 density range were demonstrated by electron microscopy. Following fast protein liquid chromatography of CSF at physiologic ionic strength, apoE was demonstrated in particles of average size greater than particles containing apoA‐I. The largest lipoproteins separated by this technique contained apoE without apoA‐I. Thus, the presence of large apoE‐containing lipoproteins was confirmed without ultracentrifugation. Interconversion between the more abundant smaller apoE‐HDL subfractions 2 and 3 and the novel larger apoE‐HDL1 is postulated to mediate a role in cholesterol redistribution in brain.
Sequential Expression of Egr‐1 and Egr‐3 in Hippocampal Granule Cells Following Electroconvulsive Stimulation
Authors: O'Donovan Kevin J. , Wilkens Eric P. , Baraban Jay M.
Published: July 2010

Abstract: Previous studies examining the regulation of immediate early gene mRNAs by neuronal stimulation have revealed that two members of the Egr family of transcription factors, Egr‐1 and Egr‐3, display parallel response patterns. As these transcription factors compete for the same consensus sequence, we investigated how their expression and DNA binding activities are coordinated. Following electroconvulsive stimulation, which induces rapid increases in both Egr‐1 and Egr‐3 mRNA levels in dentate granule cells, we found that these proteins are induced sequentially. Egr‐1 protein levels peak at 0.5–1 h and decay to basal levels by 4 h. In contrast, Egr‐3 protein levels respond more slowly; little change is apparent at 1 h, and peak levels are not reached until 4 h following stimulation. Gel shift assays demonstrated that Egr‐1 and Egr‐3 DNA binding activities follow the same pattern. These findings indicate that Egr‐1 and Egr‐3 act in concert to mediate early and late phases, respectively, of the transcriptional response regulated by their cognate response element.
Functional Characterization of Human N‐Methyl‐d‐Aspartate Subtype 1A/2D Receptors
Authors: Hess Stephen D. , Daggett Lorrie P. , Deal Charles , Lu Chin‐Chun , Johnson Edwin C. , Veliçelebi Gönül
Published: July 2010

Abstract: The human NMDAR2D subunit was cloned, and the pharmacological properties of receptors resulting from injection of transcripts encoding human NMDAR1A and NMDAR2D subunits in Xenopus oocytes were characterized by profiling NMDA receptor agonists and antagonists. We found that glutamate, NMDA, glycine, and d‐serine were significantly more potent on hNMDAR1A/2D than on hNMDAR1A/2A or hNMDAR1A/2B. Also, the potencies of NMDA and glycine were higher for hNMDAR1A/2D than for hNMDAR1A/2C. Ifenprodil was more potent at hNMDAR1A/2B than at hNMDAR1A/2D, whereas 5,7‐dichlorokynurenate was more potent at hNMDAR1A/2A than at hNMDAR1A/2D. As measured in transiently transfected human embryonic kidney 293 cells, the maximal inward current in the presence of external Mg2+ occurred at −40 mV, and full block was not observed at negative potentials. Kinetic measurements revealed that the higher affinity of hNMDAR1A/2D for both glutamate and glycine relative to hNMDAR1A/2A and hNMDA1A/2B can be explained by slower dissociation of each agonist from hNMDAR1A/2D. The hNMDAR1A/2D combination represents a pharmacologically and functionally distinct receptor subtype and may constitute a potentially important target for therapeutic agents active in the human CNS.
Ca2+‐Dependent K+ Currents Induced by Muscarinic Receptor Activation in Guinea Pig Adrenal Chromaffin Cells
Authors: Ohta Toshio , Ito Shigeo , Nakazato Yoshikazu
Published: July 2010

Abstract: The characteristics of the outward current (Iout) induced by muscarine were examined by using the whole‐cell patch‐clamp technique with a K+‐containing pipette solution in combination with fura‐2 microfluorometry in guinea pig chromaffin cells. Muscarine caused a transient increase in the cytosolic Ca2+ concentration ([Ca2+]i) and activated an Iout with a reversal potential close to a K+ equilibrium potential. Under symmetric K+ conditions, muscarine produced a transient inward current and an increase in [Ca2+]i at −60 mV. At −15 mV, apamin and charybdotoxin, respective SK and BK channel blockers, decreased the Iout but scarcely affected the [Ca2+]i response to muscarine. Muscarine produced an Iout and an increase in [Ca2+]i even after a removal of external Ca2+ and in the presence of Co2+, indicating that these responses are mediated by Ca2+ release from intracellular stores. The Iout evoked by the Ca2+ release was much smaller than that evoked by the voltage‐dependent Ca2+ influx, even when similar [Ca2+]i changes assessed by fura‐2 microfluorometry occurred. Inositol 1,4,5‐trisphosphate (InsP3) applied intracellularly and the photolysis of caged InsP3 each evoked current changes similar to those induced by muscarine. These results indicate that the Iout evoked by muscarinic stimulation is mediated by Ca2+‐dependent K+ channels (probably BK and SK channels), which are activated by Ca2+ released from intracellular Ca2+ stores in guinea pig chromaffin cells.
Isoform‐Specific Redistribution of Calcineurin Aα and Aβ in the Hippocampal CA1 Region of Gerbils After Transient Ischemia
Authors: Hashimoto Takeshi , Kawamata Toshio , Saito Naoaki , Sasaki Masahiro , Nakai Masamichi , Niu Sanyoung , Taniguchi Taizo , Terashima Akira , Yasuda Minoru , Maeda Kiyoshi , Tanaka Chikako
Published: July 2010

Abstract: To investigate isoform‐specific roles of Ca2+/calmodulin‐dependent phosphatase [calcineurin (CaN)] in ischemia‐induced cell death, we raised antibodies specific to CaN Aα and CaN Aβ and localized the CaN isoforms in the hippocampal CA1 region of Mongolian gerbils subjected to a 5‐min occlusion of carotid arteries. In the nonischemic gerbil, immunoreactions of both isoforms were highly enriched in CA1 regions, especially in the cytoplasm and apical dendrites of CA1 pyramidal neurons. At 4–7 days after the induced ischemia, immunoreactivities of the CaN Aα isoform in CA1 pyramidal cells were markedly reduced, whereas they were enhanced in the CA1 radiatum and oriens layers. In contrast, CaN Aβ immunoreactivities were reduced in all layers of the ischemic CA1 region, whereas they were enhanced in activated astrocytes, colocalizing with glial fibrillary acidic protein. These findings suggest that up‐regulation of CaN Aα in afferent fibers in CA1 and up‐regulation of CaN Aβ in reactive astrocytes may be involved in neuronal reorganization after ischemic injury.
Herpes Simplex Virus 1‐Mediated Transfer of Preproenkephalin A in Rat Dorsal Root Ganglia
Authors: Antunes Bras João Manuel , Epstein Alberto L. , Bourgoin Sylvie , Hamon Michel , Cesselin François , Pohl Michel
Published: July 2010

Abstract: Recombinant herpes simplex virus‐1 encoding the rat preproenkephalin A (HSVLatEnk1) was generated for driving the expression of preproenkephalin A‐derived peptides in dorsal root ganglia of rats in vivo. Three weeks after infection via the hind footpads, quantitative RT‐PCR and in situ hybridization experiments showed a strong expression of preproenkephalin A mRNA in lumbar dorsal root ganglia. In addition, a 40–160% increase in radioimmunoassayable Met‐enkephalin‐like material concentrations was found in the dorsal spinal cord and dorsal root ganglia, respectively, at the lumbar level in HSVLatEnk1‐infected rats as compared with animals infected with β‐galactosidase‐encoding recombinant herpes simplex virus‐1 or control rats. These data demonstrate the efficacy of the preproenkephalin A encoding vector and suggest that it should help in elucidating the role of Met‐enkephalin‐containing primary afferent fibers in pain transmission and/or control.
GM1 Ganglioside: In Vivo and In Vitro Trophic Actions on Central Neurotransmitter Systems
Authors: Hadjiconstantinou Maria , Neff Norton H.
Published: July 2010

Abstract: There is now substantial evidence that GM1 ganglioside is effective in partially correcting the consequences of neuroinjury in a number of in vivo and in vitro model systems. Although the molecular mechanism(s) and the substrates for the neurotrophic activity of the gangliosides are not fully understood, the published experimental work suggests that GM1 has antineurotoxic, neuroprotective, and neurorestorative effects on various central neurotransmitter systems. This review focuses attention on studies reporting that GM1 restores neuronal integrity and function in the brain of lesioned young as well as aged animals. Critical analysis of these studies can provide guidance for future ganglioside research and may point to novel approaches for treating neuroinjury and a variety of degenerative conditions, including aging.
Distribution of mRNAs Encoding the Peroxisome Proliferator‐Activated Receptor α, β, and γ and the Retinoid X Receptor α, β, and γ in Rat Central Nervous System
Authors: Cullingford Tim E. , Bhakoo Kishore , Peuchen Stefan , Dolphin Colin T. , Patel Ritesh , Clark John B.
Published: July 2010

Abstract: We report the isolation, by RT‐PCR, of partial cDNAs encoding the rat peroxisome proliferator‐activated receptor (PPAR) isoforms PPARα, PPARβ, and PPARγ and the rat retinoid X receptor (RXR) isoforms RXRα, RXRβ, and RXRγ. These cDNAs were used to generate antisense RNA probes to permit analysis, by the highly sensitive and discriminatory RNase protection assay, of the corresponding mRNAs in rat brain regions during development. PPARα, PPARβ, RXRα, and RXRβ mRNAs are ubiquitously present in different brain regions during development, PPARγ mRNA is essentially undetectable, and RXRγ mRNA is principally localised to cortex. We demonstrate, for the first time, the presence of PPAR and RXR mRNAs in primary cultures of neonatal meningeal fibroblasts, cerebellar granule neurons (CGNs), and cortical and cerebellar astrocytes and in primary cultures of adult cortical astrocytes. PPARα, PPARβ, RXRα, and RXRβ mRNAs are present in all cell types, albeit that PPARα and RXRα mRNAs are at levels near the limit of detection in CGNs. PPARγ mRNA is expressed at low levels in most cell types but is present at levels similar to those of PPARα mRNA in adult astrocytes. RXRγ mRNA is present either at low levels, or below the level of detection of the assay, for all cell types studied.
Cyclic AMP Protects Against Staurosporine and Wortmannin‐Induced Apoptosis and Opioid‐Enhanced Apoptosis in Both Embryonic and Immortalized (F‐11κ7) Neurons
Authors: Goswami R. , Dawson S. A. , Dawson G.
Published: July 2010

Abstract: The mechanism by which opiates affect fetal development is unknown, but one potential target is the programmed cell death (apoptosis) pathway of neurons. Apoptosis was induced in both primary neuronal cultures from embryonic day 7 cerebral hemispheres of chick brain (E7CH) and the F‐11κ7 cell line (an immortalized mouse neuroblastoma × dorsal root ganglion hybrid stably transfected to overexpress κ‐opioid receptors) by either staurosporine or the phosphatidylinositol 3‐kinase inhibitors wortmannin and LY294002. Cells pretreated with either the μ‐specific opioid agonist morphiceptin (E7CH) or the κ‐specific opioid agonist U69,593 (F‐11κ7) for 24 h showed increased apoptosis in response to staurosporine or wortmannin when compared with nonpretreated cells. The effects of morphiceptin and U69,593 were time‐ and dose‐dependent and antagonist‐reversible, suggesting that they were receptor‐mediated. Neither morphiceptin nor U69,593 by themselves had any measurable effect on cell viability or DNA fragmentation, and coaddition of opiates at the same time as staurosporine, wortmannin, or LY294002 did not enhance apoptosis. Time course studies indicated a maximal opioid effect at a time (16–24 h) when inhibition of adenylate cyclase had been maximal for many hours. Addition of dibutyryl cyclic AMP either before or at the time of opioid addition protected against apoptosis and reduced fragmentation to levels seen for staurosporine plus dibutyryl cyclic AMP alone. The specificity for cyclic AMP was confirmed by showing protection with the specific agonist Sp‐adenosine 3′,5′‐cyclic monophosphothioate and increased killing with the antagonist Rp‐adenosine 3′,5′‐cyclic monophosphothioate. We conclude that the opioid enhancement of apoptosis is based on the inhibition of adenylate cyclase and that the effect is time‐dependent.
Cytotoxic Effect of β‐Amyloid on a Human Differentiated Neuron Is Not Mediated by Cytoplasmic Ca2+ Accumulation
Authors: Gao Zhi‐yong , Collins Heather W. , Matschinsky Franz M. , Lee Virginia M.‐Y. , Wolf Bryan A.
Published: July 2010

Abstract: The effects of synthetic β‐amyloid (Aβ1–42) on cell viability and cellular Ca2+ homeostasis have been studied in the human neuron‐like NT2N cell, which differentiates from a teratocarcinoma cell line, NTera2/C1.D1, by retinoic acid treatment. NT2N viability was measured using morphological criteria and fluorescent live/dead staining and quantified using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide metabolism. Aβ1–42 dose‐dependently caused NT2N cell death when it was present in the cell culture for 14 days but had no effect on viability when it was present for 4 days. The lowest effective concentration was 4 µM, and the strongest effect was produced by 40 µM. Control NT2N cells produced spontaneous cytosolic Ca2+ oscillations under basal conditions. These oscillations were inhibited dose‐dependently (0.4–40 µM) by Aβ1–42 that was present in the cell culture for 1 or 4 days. Ca2+ wave frequency was decreased from 0.21 ± 0.02 to 0.05 ± 0.02/min, amplitude from 88 ± 8 to 13 ± 4 nM, and average Ca2+ level from 130 ± 8 to 58 ± 3 nM. The Ca2+ responses to 30 mM K+ and 100 µM glutamate were not different between control and Aβ‐treated cells. Thus, the results do not support the hypothesis that cytosolic early Ca2+ accumulation mediates Aβ‐induced NT2N cell death.
Apoptosis of Central and Peripheral Neurons Can Be Prevented with Cyclin‐Dependent Kinase/Mitogen‐Activated Protein Kinase Inhibitors
Authors: Maas James W. , Horstmann Sonja , Borasio Gian Domenico , Anneser Johanna M. H. , Shooter Eric M. , Kahle Philipp J.
Published: July 2010

Abstract: Previous studies have indicated that certain members of the cyclin‐dependent kinase/mitogen‐activated protein kinase superfamily are involved in apoptosis of neuronal cells. Here, we have examined programmed cell death induced by withdrawal of neurotrophic support from CNS (rat retinal) and PNS (chick sympathetic, sensory, and ciliary) neurons. All four neuron types were equally rescued by the purine analogues olomoucine and roscovitine. Olomoucine inhibits multiple cyclin‐dependent and mitogen‐activated protein kinases with similar potency. Roscovitine is a more selective cyclin‐dependent kinase inhibitor; but, so is butyrolactone I, which did not prevent retinal ganglion cell death. The specific p38MAPK inhibitor SB‐203580 did not prevent apoptosis in retinal ganglion cells. Death of these cells in the absence of neurotrophic factors was accompanied by morphological changes indicative of apoptosis, including nuclear condensation and fragmentation. Treatment with olomoucine or roscovitine not only prevented these apoptotic changes in retinal ganglion cells but also blocked neurite outgrowth. The survival‐promoting activity of olomoucine correlated with its in vitro IC50 for c‐Jun N‐terminal kinase‐1 and its potency to repress c‐jun induction in live PC12 cells. Roscovitine was more potent in rescuing neurons than in inhibiting Jun kinase. Thus, the antiapoptotic action of roscovitine might be due to inhibition of additional kinases.
Cyclic AMP Resets the Circadian Clock in Cultured Xenopus Retinal Photoreceptor Layers
Authors: Hasegawa Minoru , Cahill Gregory M.
Published: July 2010

Abstract: The Xenopus retinal photoreceptor layer contains a circadian oscillator that regulates melatonin synthesis in vitro. The phase of this oscillator can be reset by light or dopamine. The phase‐response curves for light and dopamine are similar, with transitions from phase delays to phase advances in the mid‐subjective night. Light and dopamine each can inhibit adenylate cyclase in retinal photoreceptors, suggesting cyclic AMP as a candidate second messenger for entrainment of the circadian oscillator. We report here that treatments that increase intracellular cyclic AMP reset the phase of the photoreceptor circadian oscillator, and that the phase‐response curves for these treatments are 180° out of phase with the phase‐response curves for light and dopamine. Activation of adenylate cyclase by forskolin during the late subjective day or early subjective night caused phase advances. The same treatment during the late subjective night or early subjective day caused phase delays. Similar phase shifts were induced by 3‐isobutyl‐1‐methyl‐xanthine (a phosphodiesterase inhibitor) or 8‐(4‐chlorophenylthio)cyclic AMP. All of these treatments also acutely increased melatonin release. Forskolin and 3‐isobutyl‐1‐methylxanthine increased the accumulation of intracellular cyclic AMP, but not cyclic GMP, in photoreceptor layers. The results indicate that cyclic AMP‐dependent pathways regulate the photoreceptor circadian oscillator and suggest that a decrease in cyclic AMP may be involved in circadian entrainment by light and/or dopamine.
Nitric Oxide Causes Glutamate Release from Brain Synaptosomes
Authors: McNaught Kevin St. P. , Brown Guy C.
Published: July 2010

Abstract: We determined the ability of pathological levels of nitric oxide (NO) to cause glutamate release from isolated rat brain nerve terminals using a fluorometric assay. It was found that NO (0.7 and 2 µM) produced (4 and 10 nmol/mg of synaptosomal protein) Ca2+‐independent glutamate release from synaptosomes (after 1 min of exposure). Spermine/NO complex (spermine NONOate; a slow NO donor) and potassium cyanide (an inhibitor of cytochrome oxidase) also caused Ca2+‐independent glutamate release. Preincubation of synaptosomes with 5 µM 1H‐[1,2,4]oxadiazole[4,3‐a]quinoxalin‐1‐one (an inhibitor of soluble guanylyl cyclase) had no effect on NO‐induced Ca2+‐independent glutamate release. Ca2+‐independent glutamate release produced by NO was greater in a low‐oxygen medium. NO, spermine NONOate, and potassium cyanide inhibited synaptosomal respiration with a similar order of potency with respect to their ability to cause glutamate release. Because NO has been shown previously to inhibit reversibly cytochrome oxidase in competition with oxygen, our findings in this study suggest that NO (and cyanide) causes glutamate release following inhibition of mitochondrial respiration at the level of cytochrome oxidase. Thus, elevated NO production leading to mitochondrial dysfunction, glutamate release, and excitotoxicity may contribute to neuronal death in neurological diseases.
Lipopolysaccharide Intranigral Injection Induces Inflammatory Reaction and Damage in Nigrostriatal Dopaminergic System
Authors: Castaño A. , Herrera A. J. , Cano J. , Machado A.
Published: July 2010

Abstract: The pathogenesis of Parkinson's disease is still poorly understood. To address the hypothesis that immunemediated events, such as microglial activation, may be involved in the dopaminergic neurodegeneration, we have studied the effect that intranigral injection of the immunostimulant lipopolysaccharide has on monoaminergic neurotransmitters in rats. Activation of microglial cells, visualized by immunohistochemistry with a specific monoclonal antibody, was already obvious 2 days after injection. In relation to the biochemical parameters studied, we found a significant decrease of dopamine levels in both the substantia nigra and striatum up to at least 21 days after intranigral injection of lipopolysaccharide. This result was supported by the decrease in tyrosine hydroxylase activity and the loss of tyrosine hydroxylase‐positive neuronal bodies, shown by immunohistochemistry. These alterations of the dopaminergic system did not reverse during the interval studied (21 days); conversely, the serotoninergic system suffered only transient damage. In addition, we found that the neurotoxic effect of lipopolysaccharide was not mediated by nitric oxide. Based on our results we suggest that the nigrostriatal dopaminergic system is susceptible to damage by inflammatory events and that these may be implicated in neurodegeneration processes such as Parkinson's disease.
Chronic Exposure to Aluminum Impairs Neuronal Glutamate‐Nitric Oxide‐Cyclic GMP Pathway
Authors: Cucarella Carmen , Montoliu Carmina , Hermenegildo Carlos , Sáez Rosana , Manzo Luigi , Miñana María‐Dolores , Felipo Vicente
Published: July 2010

Abstract: Humans are exposed to aluminum from environmental sources and therapeutic treatments. However, aluminum is neurotoxic and is considered a possible etiologic factor in Alzheimer's disease and other neurological disorders. The molecular mechanism of aluminum neurotoxicity is not understood. We tested the effects of aluminum on the glutamate‐nitric oxide‐cyclic GMP pathway in cultured neurons. Neurons were exposed to 50 µM aluminum in culture medium for short‐term (4 h) or long‐term (8–14 days) periods, or rats were prenatally exposed, i.e., 3.7% aluminum sulfate in the drinking water, during gestation. Chronic (but not short‐term) exposure of neurons to aluminum decreased glutamate‐induced activation of nitric oxide synthase by 38% and the formation of cyclic GMP by 77%. The formation of cyclic GMP induced by the nitric oxide‐generating agent S‐nitroso‐N‐acetylpenicillamine was reduced by 33%. In neurons from rats prenatally exposed to aluminum but not exposed to it during culture, glutamate‐induced formation of cyclic GMP was inhibited by 81%, and activation of nitric oxide synthase was decreased by 85%. The formation of cyclic GMP induced by S‐nitroso‐N‐acetylpenicillamine was not affected. These results indicate that chronic exposure to aluminum impairs glutamate‐induced activation of nitric oxide synthase and nitric oxide‐induced activation of guanylate cyclase. Impairment of the glutamate‐nitric oxide‐cyclic GMP pathway in neurons may contribute to aluminum neurotoxicity.
Differential Cleavage of Provasopressin by the Major Molecular Forms of SPC3
Authors: Coates Leigh C. , Birch Nigel P.
Published: July 2010

Abstract: We have investigated the roles of full‐length and carboxyl‐terminus‐truncated forms of the subtilisin‐like prohormone convertase SPC3 in the processing of the radiolabeled vasopressin and oxytocin precursors, in vitro. We found SPC3 cleaves provasopressin at both the vasopressin‐neurophysin and neurophysin‐glycopeptide processing sites. Prooxytocin is cleaved by SPC3 at the oxytocin‐neurophysin cleavage site. However, our results reveal differences in processing of provasopressin by the different molecular forms of SPC3. In incubations where the rate of autocatalytic carboxyl‐terminus truncation of SPC3 was dramatically reduced, 86‐kDa SPC3, which has an unprocessed carboxyl terminus, cleaved provasopressin at the neurophysin‐glycopeptide junction. Cleavage at the vasopressin‐neurophysin junction only occurred with the appearance of carboxyl‐terminus‐truncated forms of the enzyme. Incubations containing 64‐kDa SPC3 or 64‐kDa SPC3‐T, a recombinant form of SPC3 truncated 14 amino acids beyond the conserved carboxyl‐terminal “P‐domain,” rapidly cleaved provasopressin at both the vasopressin‐neurophysin and neurophysin‐glycopeptide junctions. Our results also suggest that prooxytocin is unable to be cleaved by the 86‐kDa form of SPC3. We propose that SPC3 should be considered as a candidate endoprotease in the biosynthesis of vasopressin. Furthermore, we suggest that the carboxyl terminus of SPC3 alters the cleavage specificity of SPC3.
Structure‐Activity Relationships of Conformationally Constrained Peptide Analogues of Loop 2 of Brain‐Derived Neurotrophic Factor
Authors: O'Leary P. D. , Hughes R. A.
Published: July 2010

Abstract: Brain‐derived neurotrophic factor (BDNF) promotes the survival of various neuronal populations and thus shows potential in the treatment of neurodegenerative disease. However, BDNF is not pharmacokinetically optimal for use as a therapeutic agent. As a step toward the development of low‐molecular‐weight BDNF‐like drugs, we have designed a series of small, conformationally constrained peptides of various sizes using the three‐dimensional structure of BDNF derived by homology modeling as a template. When tested in cultures of embryonic chick sensory neurons the peptides produced concentration‐dependent inhibition of BDNF‐mediated neuronal survival and caused both a rightward shift and depression of the maximum of the BDNF concentration‐response curve. The compounds had no effect on the survival response to nerve growth factor and were without intrinsic trophic or toxic effects when added to cultures alone. With the aid of pharmacodynamic simulations we demonstrated that the inhibitory activity of the active peptides is consistent with them acting as competitive antagonists of BDNF for its high‐affinity receptor, trkB. An alanine scan of the largest peptide identified several residues important in mediating the inhibitory action of the peptides. We intend to use the data from these studies to develop small peptidic BDNF‐like agonists.
Somatostatin Potently Stimulates In Vivo Striatal Dopamine and γ‐Aminobutyric Acid Release by a Glutamate‐Dependent Action
Authors: Hathway G. J. , Emson P. C. , Humphrey P. P. A. , Kendrick K. M.
Published: July 2010

Abstract: We have used in vivo microdialysis in anaesthetised rats to investigate whether somatostatin (SRIF) can play a neuromodulatory role in the striatum. When 100 nM SRIF was retrodialysed for 15 min, it increased concentrations of dopamine (DA) by 28‐fold, γ‐aminobutyric acid (GABA) by eightfold, and glutamate (Glu) by sixfold as well as those of aspartate (Asp) and taurine (Tau). These effects were both calcium‐ and tetrodotoxin‐sensitive. Lower (10 or 50 nM) and higher (1 µM) SRIF concentrations were less effective. Rapid sampling showed that whereas Asp and Glu concentrations were raised for 3 min at the start of 15‐min SRIF infusions, those of DA were increased for 12 min. A second 15‐min application of 100 nM SRIF given 135 min after the first application failed to increase transmitter release. An NMDA receptor antagonist, 2‐amino‐5‐phosphonopentanoic acid (200 µM), blocked SRIF (100 nM)‐evoked Asp, Glu, Tau, and GABA release and reduced that of DA. An α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA)/kainate antagonist, 6,7‐dinitroquinoxaline‐2,3‐dione (100 µM), blocked SRIF‐induced DA and Tau release and reduced that of Asp, Glu, and GABA. These results show that SRIF increases DA, Glu, Asp, GABA, and Tau release in the rat striatum and suggest that its actions on DA and GABA release are mainly mediated through increased excitatory amino acid release.
Activation of p38MAPK in Microglia After Ischemia
Authors: Walton Kevin M. , DiRocco Richard , Bartlett Becky A. , Koury Elizabeth , Marcy Val Robert , Jarvis Bruce , Schaefer Erik M. , Bhat Ratan V.
Published: July 2010

Abstract: p38MAPK has been implicated in the regulation of proinflammatory cytokines and apoptosis in vitro. To understand its role in neurodegeneration, we determined the time course and localization of the dually phosphorylated active form of p38MAPK in hippocampus after global forebrain ischemia. Phosphorylated p38MAPK and mitogen‐activated protein kinase‐activated protein 2 activity increased over 4 days after ischemia. Phosphorylated p38MAPK immunoreactivity was observed in microglia in regions adjacent to, but not in, the dying CA1 neurons. In contrast, neither c‐Jun N‐terminal kinase 1 nor p42/p44MAPK activity was altered after ischemia. These results provide the first evidence for localization of activated p38MAPK in the CNS and support a role for p38MAPK in the microglial response to stress.