bluuebunny/crossref_metadata_2025 huggingface dataset:
Age‐Related Differences in the Effect of Dichloroacetate on Postischemic Lactate and Acid Clearance Measured In Vivo Using Magnetic Resonance Spectroscopy and Microdialysis
Authors: Corbett Ron , Laptook Abbot , Gee Jerry , Garcia Damian , Silmon Sandra , Tollefsbol Greg
Published: July 2010

Abstract: Numerous studies using adult animal models suggest that dichloroacetate (DCA) may have neuroprotective properties by virtue of its ability to increase rates of metabolism and, therefore, clearance of brain lactic acidosis, which may accumulate during cerebral ischemia. We tested the hypothesis that postischemic DCA administration affects lactate and acid clearance to different extents in immature versus mature brain. 31P and 1H magnetic resonance spectroscopy were used to measure intracellular acid and lactate clearance rates in vivo in newborn and 1‐month‐old swine after a 14‐min episode of transient near‐complete global ischemia. Simultaneous monitoring of extracellular lactate efflux and clearance was measured in the same animals by in vivo microdialysis. Plasma glucose concentrations were elevated in order to study animals with severe cerebral lactic acidosis. Maximal levels of brain lactosis (16–20 µmol/g) and acidosis (pHintracellular 5.8–6.0) were reached during the first 10 min of recovery and were the same in age groups and in subgroups either acting as controls or treated with DCA (200 mg/kg) given from the last minute of ischemia to 5–7 min after ischemia. For newborns, DCA administration improved the postischemic clearance rate of cerebral acidosis and cerebral phosphocreatine, with similar trends for the clearance of lactosis and increased rates of recovery of nucleotide triphosphates, compared with controls. In contrast, DCA administration in 1‐month‐olds resulted in a modest trend for improvement of cerebral lactate clearance, but did not affect acid clearance or the recovery rate of phosphocreatine or nucleotide triphosphates. Extracellular brain lactate concentrations had similar relative increases and rates of decline for subgroups of either age treated with DCA versus controls. The results of this study indicate that postischemic DCA administration helps to resolve cerebral acidosis to a greater degree in immature than more mature brain, suggesting that DCA may have cerebroprotective properties for neonatal hypoxic‐ischemic encephalopathy.
Hypoxic Preconditioning and Hypoxic‐Ischemic Brain Damage in the Immature Rat: Pathologic and Metabolic Correlates
Authors: Vannucci Robert C. , Towfighi Javad , Vannucci Susan J.
Published: July 2010

Abstract: It has been reported that immature rats subjected to cerebral hypoxia‐ischemia sustain less brain damage if they are previously exposed to systemic hypoxia compared with animals not exposed to prior hypoxia. Accordingly, neuropathologic and metabolic experiments were conducted to confirm and extend the observation that hypoxic preconditioning protects the perinatal brain from subsequent hypoxic‐ischemic brain damage. Six‐day postnatal rats were subjected to systemic hypoxia with 8% oxygen at 37°C for 2.5 h. Twenty‐four hours later, they were exposed to unilateral cerebral hypoxia‐ischemia for 2.5 h, produced by unilateral common carotid artery ligation and systemic hypoxia with 8% oxygen. Neuropathologic analysis, conducted at 30 days of postnatal age, indicated a substantial reduction in the severity of brain damage in the preconditioned rats, such that only 6 of 14 such animals exhibited cystic infarction, but all 13 animals without prior preconditioning exhibited infarction (p < 0.001). Measurement of cerebral glycolytic and tricarboxylic acid intermediates and high‐energy phosphate reserves at the terminus of and at 4 and 24 h following hypoxia‐ischemia showed no differences in the extent of alterations in the preconditioned and nonpreconditioned immature rats. A difference was seen in the restitution of high‐energy stores during the first 24 h of recovery from hypoxia‐ischemia, with a more optimal preservation of these metabolites in the preconditioned animals, reflecting the less severe ultimate brain damage. Accordingly, the neuroprotection afforded to the preconditioned animals was not the result of any differences in the extent of anaerobic glycolysis, tissue acidosis, or depletion in high‐energy reserves during hypoxia‐ischemia but rather the result of other mechanisms that improved the metabolic status of the immature brain during the early hours of reperfusion following hypoxia‐ischemia.
Inhibition of Voltage‐Sensitive Calcium Channels by the A2A Adenosine Receptor in PC12 cells
Authors: Park Tae‐Ju , Chung Sungkwon , Han Myung Kwan , Kim Uh‐Hyun , Kim Kyong‐Tai
Published: July 2010

Abstract: The role of the A2A adenosine receptor in regulating voltage‐sensitive calcium channels (VSCCs) was investigated in PC12 cells. Ca2+ influx induced by membrane depolarization with 70 mM K+ could be inhibited with CGS21680, an A2A receptor‐specific agonist. Both L‐ and N‐type VSCCs were inhibited by CGS21680 treatment. Effects of adenosine receptor agonists and antagonists indicate that the typical A2A receptor mediates inhibition of VSCCs. Cholera toxin (CTX) treatment for 24 h completely eliminated the CGS21680 potency. Similar inhibitory effects on VSCCs were obtained by membrane‐permeable activators of protein kinase A (PKA). These effects were blocked by Rp‐adenosine‐3′,5′‐cyclic monophosphothioate, a PKA inhibitor. The data suggest that activation of the A2A receptor leads to inhibition of VSCCs via a CTX‐sensitive G protein and PKA. ATP pretreatment caused a reduction in subsequent rise in cytosolic free Ca2+ concentration induced by 70 mM K+, presumably by inactivation of VSCCs. Simultaneous treatment with ATP and CGS21680 produced significantly greater inhibition of VSCCs than treatment with CGS21680 or ATP alone. Furthermore, the CGS21680‐induced inhibition of VSCCs was not affected by the presence of reactive blue 2. CGS21680 still significantly inhibited ATP‐evoked Ca2+ influx without VSCC activity after cobalt or 70 mM K+ pretreatment. These data suggest that the A2A receptor‐sensitive VSCCs differ from those activated by ATP treatment. Although A2A receptors induce inhibition of VSCCs as well as ATP‐induced Ca2+ influx, the two inhibitory effects are clearly distinct from each other.
Expression and Transcriptional Regulation of the Human α3 Neuronal Nicotinic Receptor Subunit in T Lymphocyte Cell Lines
Authors: Battaglioli Elena , Gotti Cecilia , Terzano Susanna , Flora Adriano , Clementi Francesco , Fornasari Diego
Published: July 2010

Abstract: The expression of neurotransmitter receptors on the surface of immunocompetent cells is generally accepted as evidence that the nervous system can influence immune responses, even though many aspects of these interactions remain to be elucidated. In this article, we analyzed the expression of the α3 nicotinic receptor subunit in human cell lines of myeloid and lymphoid origin and show that the α3 mRNA and the receptor molecules containing this subunit are specifically expressed in T lymphocyte cell lines. We have previously characterized the structural properties of the human α3 nicotinic subunit gene promoter and defined its functional profile in neuronal cells; in this study, we analyzed the activity of the α3 promoter in T lymphocytes and found that the same minimal promoter located in the 0.16‐kb BglII‐AccIII fragment is responsible for the expression of the α3 mRNA in both neuronal and T lymphocyte cell lines. However, the α3 transcription initiation patterns in the two cell types were both qualitatively and quantitatively different, and the minimal promoter was differentially modulated by downstream and upstream regulatory elements. These findings suggest that distinct transcriptional mechanisms allow the same promoter to be regulated in a tissue‐specific fashion, according to the different functional needs of the two cell types.
Endothelin Induces a Calcium‐Dependent Phosphorylation of PEA‐15 in Intact Astrocytes: Identification of Ser104 and Ser116 Phosphorylated, Respectively, by Protein Kinase C and Calcium/Calmodulin Kinase II In Vitro
Authors: Kubes Miroslav , Cordier Jocelyne , Glowinski Jacques , Girault Jean‐Antoine , Chneiweiss Hervé
Published: July 2010

Abstract: PEA‐15 (phosphoprotein enriched in astrocytes, Mr = 15,000) is an acidic serine‐phosphorylated protein highly expressed in the CNS, where it can play a protective role against cytokine‐induced apoptosis. PEA‐15 is a major substrate for protein kinase C. Endothelins, which are known to exert pleiotropic effects on astrocytes, were used to analyze further the processes involved in PEA‐15 phosphorylation. Endothelin‐1 or endothelin‐3 (0.1 µM) induced a robust phosphorylation of PEA‐15 that was abolished by the removal of extracellular calcium, but only diminished by inhibitors of protein kinase C. Microsequencing of phosphopeptides generated by digestion of PEA‐15 following endothelin‐1 treatment identified two phosphorylated residues: Ser104, previously recognized as the protein kinase C site, and a novel phosphoserine, Ser116, located in a consensus motif for either protein kinase casein kinase II or calcium/calmodulin‐dependent protein kinase II (CaMKII). Partly purified PEA‐15 was a substrate in vitro for CaMKII, but not for casein kinase II. Two‐dimensional phosphopeptide mapping demonstrated that the site phosphorylated in vitro by CaMKII was also phosphorylated in intact astrocytes in response to endothelin. CaMKII phosphorylated selectively Ser116 and had no effect on Ser104, but in vitro phosphorylation by CaMKII appeared to facilitate further phosphorylation by protein kinase C. Treatment of intact astrocytes with okadaic acid enhanced the phosphorylation of the CaMKII site. These results demonstrate that PEA‐15 is phosphorylated in astrocytes by CaMKII (or a related kinase) and by protein kinase C in response to endothelin.
Compartmentation of [14C]Glutamate and [14C]Glutamine Oxidative Metabolism in the Rat Hippocampus as Determined by Microdialysis
Authors: Zielke H. Ronald , Collins Roger M. , Baab Peter J. , Huang Yinyin , Zielke Carol L. , Tildon J. Tyson
Published: July 2010

Abstract: Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free‐moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect 14CO2 generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min‐fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced 14C‐amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [14C]glutamate and [14C]glutamine were 59 ± 18 and 2.1 ± 0.5 dpm/pmol, respectively. The initial infused specific activities for [U‐14C]glutamate and [U‐14C]glutamine were 408 ± 8 and 387 ± 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [14C]glutamine in the interstitial space when [14C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.
Differential Regulation of Focal Adhesion Kinase and Mitogen‐Activated Protein Kinase Tyrosine Phosphorylation During Insulin‐Like Growth Factor‐I‐Mediated Cytoskeletal Reorganization
Authors: Kim Bhumsoo , Feldman Eva L.
Published: July 2010

Abstract: In SH‐SY5Y human neuroblastoma cells, insulin‐like growth factor (IGF)‐I mediates membrane ruffling and growth cone extension. We have previously shown that IGF‐I activates the tyrosine phosphorylation of focal adhesion kinase (FAK) and extracellular signal‐regulated protein kinase (ERK) 2. In the current study, we examined which signaling pathway underlies IGF‐I‐mediated FAK phosphorylation and cytoskeletal changes and determined if an intact cytoskeleton was required for IGF‐I signaling. Treatment of SH‐SY5Y cells with cytochalasin D disrupted the actin cytoskeleton and prevented any morphological changes induced by IGF‐I. Inhibitors of phosphatidylinositol 3‐kinase (PI 3‐K) blocked IGF‐I‐mediated changes in the actin cytoskeleton as measured by membrane ruffling. In contrast, PD98059, a selective inhibitor of ERK kinase, had no effect on IGF‐I‐induced membrane ruffling. In parallel with effects on the actin cytoskeleton, cytochalasin D and PI 3‐K inhibitors blocked IGF‐I‐induced FAK tyrosine phosphorylation, whereas PD98059 had no effect. It is interesting that cytochalasin D did not block IGF‐I‐induced ERK2 tyrosine phosphorylation. Therefore, it is likely that FAK and ERK2 tyrosine phosphorylations are regulated by separate pathways during IGF‐I signaling. Our study suggests that integrity as well as dynamic motility of the actin cytoskeleton mediated by PI 3‐K is required for IGF‐I‐induced FAK tyrosine phosphorylation, but not for ERK2 activation.
The Making of Neurexins
Authors: Missler Markus , Fernandez‐Chacon Rafael , Südhof Thomas C.
Published: July 2010

Abstract: Neurexins are neuronal cell‐surface proteins with up to thousands of isoforms. These isoforms are generated by alternative splicing of transcripts from six promoters in three genes. The structure of neurexins resembles cell‐surface receptors with a modular architecture suggestive of a sequential assembly during evolution. Neurexins probably perform multiple functions in the brain. They participate in intercellular junctions in which β‐neurexins tightly bind to a second class of neuronal cell‐surface receptors called neuroligins. Intracellularly, the neurexin/neuroligin junction is bound by CASK on the neurexin side and PSD95 on the neuroligin side. CASK and PSD95 are homologous membrane‐associated guanylate kinases that bind to the neurexin/neuroligin junction via PDZ domains, creating an asymmetric junction (neurexin/neuroligin) with similar intracellular binding partners. In addition to a function as cell‐adhesion molecules, neurexins may also serve as a signalling receptor, because a class of ligands for α‐neurexins called neurexophilins is similar to peptide hormones. Finally, at least one neurexin isoform, neurexin Iα, represents a high‐affinity receptor for α‐latrotoxin, which is a potent excitatory neurotoxin. Thus, neurexins constitute a large family of neuronal receptors that may be involved in multiple interactive functions between neurons.
S100β Increases Levels of β‐Amyloid Precursor Protein and Its Encoding mRNA in Rat Neuronal Cultures
Authors: Li Yuekui , Wang Jianzhong , Sheng Jin G. , Liu Ling , Barger Steven W. , Jones Richard A. , Van Eldik Linda J. , Mrak Robert E. , Griffin W. Sue T.
Published: July 2010

Abstract: S100β has been implicated in the formation of dystrophic neurites, overexpressing β‐amyloid precursor protein (βAPP), in the β‐amyloid plaques of Alzheimer's disease. We assessed the effects of S100β on cell viability of, neurite outgrowth from, and βAPP expression by neurons in primary cultures from fetal rat cortex. S100β (1–10 ng/ml) enhanced neuronal viability (as assessed by increased mitochondrial activity and decreased lactic acid dehydrogenase release) and promoted neurite outgrowth. Higher levels of S100β (100 ng/ml, but not 1 µg/ml) produced qualitatively similar, but less marked, effects. S100β also induced increased neuronal expression of the microtubule‐associated protein MAP2, an effect that is consistent with trophic effects of S100β on neurite outgrowth. S100β (10 and 100 ng/ml) induced graded increases in neuronal expression of βAPP and of βAPP mRNA. These results support our previous suggestion that excessive expression of S100β by activated, plaque‐associated astrocytes in Alzheimer's disease contributes to the appearance of dystrophic neurites overexpressing βAPP in diffuse amyloid deposits, and thus to the conversion of these deposits into the diagnostic neuritic β‐amyloid plaques.
Interleukin‐1β and Tumor Necrosis Factor‐α Suppress Dexamethasone Induction of Glutamine Synthetase in Primary Mouse Astrocytes
Authors: Huang Tina L. , O'Banion M. Kerry
Published: July 2010

Abstract: Astrocytes play a key role in the protection of neurons from excitotoxicity by taking up excess glutamate and converting it to glutamine via the enzyme glutamine synthetase. In a number of cell types, glucocorticoid hormones induce glutamine synthetase. Glucocorticoids also down‐regulate many genes induced by proinflammatory cytokines. As the glucocorticoid receptor has been shown to interact with transcription factors that may also be activated by the proinflammatory cytokines interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α), we hypothesized that IL‐1β or TNF‐α might oppose the induction of glutamine synthetase by dexamethasone. Primary mouse cortical astrocytes were treated with 10−7M dexamethasone and doses of IL‐1β or TNF‐α ranging from 0.02 to 5 ng/ml or 0.05 to 20 ng/ml, respectively. We found that both cytokines attenuated the dexamethasone induction of glutamine synthetase protein at 24 h and that the effect was dose‐dependent. We also found that IL‐1β and TNF‐α inhibited the induction of glutamine synthetase mRNA by dexamethasone, and that the induction of enzymatic activity was similarly prevented by IL‐1β. As glutamine synthetase can be induced by physiological levels of glucocorticoids, the release of proinflammatory cytokines following acute injury or in neurodegenerative disorders may hinder the ability of astrocytes to protect neurons from excitotoxicity.
[3H]Homoquinolinate Binds to a Subpopulation of NMDA Receptors and to a Novel Binding Site
Authors: Brown J. C. , Tse H. W. , Skifter D. A. , Christie J. M. , Andaloro V. J. , Kemp M. C. , Watkins J. C. , Jane D. E. , Monaghan D. T.
Published: July 2010

Abstract: NMDA receptors mediate several important functions in the CNS; however, little is known about the pharmacology, biochemistry, and function of distinct NMDA receptor subtypes in brain tissue. To facilitate the study of native NMDA receptor subpopulations, we have determined the radioligand binding properties of [3H]homoquinolinate, a potential subtype‐selective ligand. Using quantitative receptor autoradiography, NMDA‐specific [3H]homoquinolinate binding selectively labeled brain regions expressing NR2B mRNA (layers I–III of cerebral cortex, striatum, hippocampus, and septum). NMDA‐specific [3H]homoquinolinate binding was low in brain regions that express NR2C and NR2D mRNA (cerebellar granular cell layer, NR2C; glomerular layer of olfactory bulb, NR2C/NR2D; and midline thalamic nuclei, NR2D). In forebrain, the pattern of NMDA‐specific [3H]homoquinolinate binding paralleled NR2B and not NR2A distribution. In addition to NMDA‐displaceable binding, there was a subpopulation of [3H]homoquinolinate binding sites in the forebrain, cerebellum, and choroid plexus that was not displaced by NMDA or l‐glutamate. In contrast, we found that the derivative of homoquinolinate, 2‐carboxy‐3‐carboxymethylquinoline, markedly inhibited the NMDA‐insensitive binding of [3H]homoquinolinate without inhibiting the NMDA‐sensitive population. [3H]Homoquinolinate may be useful for selectively characterizing NR2B‐containing NMDA receptors in a preparation containing multiple receptor subtypes and for characterizing a novel binding site of unknown function.
Opposing Contributions of NR1 and NR2 to Protein Kinase C Modulation of NMDA Receptors
Authors: Grant Elfrida R. , Bacskai Brian J. , Anegawa Norifusa J. , Pleasure David E. , Lynch David R.
Published: July 2010

Abstract: N‐Methyl‐d‐aspartate (NMDA) receptors mediate increases in intracellular calcium that can be modulated by protein kinase C (PKC). As PKC modulation of NMDA receptors in neurons is complex, we studied the effects of PKC activation on recombinant NMDA receptor‐mediated calcium rises in a nonneuronal mammalian cell line, human embryonic kidney 293 (HEK‐293). Phorbol 12‐myristate 13‐acetate (PMA) pretreatment of HEK‐293 cells enhanced or suppressed NMDA receptor‐mediated calcium rises based on the NMDA receptor subunit composition. NR2A or NR2B, in combination with NR1011, conveyed enhancement whereas NR2C and NR2D conveyed suppression. The PKC inhibitor bisindolylmaleimide blocked each of these effects. The region on NR2A that conveyed enhancement localized to a discrete segment of the C terminus distal to the portion of NR2C that is homologous to NR2A. Calcium‐45 accumulation, but not intracellular calcium store depletion, matched PMA effects on NMDA receptor‐mediated calcium changes, suggesting that these effects were not due to effects on intracellular calcium stores. The suppression of intracellular calcium transients seen with NR2C was eliminated when combined with NR1 splice variants lacking C‐terminal cassette 1. Thus, the intracellular calcium effects of PMA were distinguishable based on both the NR1 splice variant and the NR2 subunit type that were expressed. Such differential effects resemble the diversity of PKC effects on NMDA receptors in neurons.
Mechanisms for Facilitation of Nitric Oxide‐Evoked [3H]GABA Release by Removal of Hydroxyl Radical
Authors: Ohkuma Seitaro , Katsura Masashi , Hibino Yasushi , Hara Atsuo , Shirotani Kenji , Ishikawa Emiko , Kuriyama Kinya
Published: July 2010

Abstract: We have investigated the mechanisms for enhancement of nitric oxide (NO)‐evoked γ‐[3H]aminobutyric acid ([3H]GABA) release from mouse cerebro‐cortical neurons by hydroxyl radical (•OH) scavengers. •OH scavengers, such as N,N′‐dimethylthiourea (DMTU), uric acid, and mannitol, dose‐dependently facilitated NO‐evoked [3H]GABA release evoked by NO liberated from S‐nitroso‐N‐acetylpenicillamine. Ionomycin‐evoked [3H]GABA release, which was significantly inhibited by hemoglobin and an NO synthase, NG‐methyl‐l‐arginine, was also enhanced by DMTU. These results indicate that GABA release evoked by both endogenous and exogenous NO is facilitated by •OH scavengers. These enhancing actions of •OH scavengers were completely abolished by Ca2+ removal from incubation buffer and by an l‐type voltage‐dependent Ca2+ channel (VDCC) inhibitor, nifedipine, whereas each •OH scavenger showed no effects on [3H]GABA release in the absence of NO. Inhibitors for P/Q‐ and N‐type VDCCs had no effects on the enhancement. NO‐induced 45Ca2+ influx was also dose‐dependently enhanced by •OH scavengers, although 45Ca2+ influx was not altered by •OH scavengers in the absence of NO. Nifedipine abolished this enhancement of the NO‐induced 45Ca2+ influx by •OH scavengers. These results indicate that the removal of •OH by its scavengers facilitates the NO‐evoked [3H]GABA release dependent on Ca2+ and that this enhancement is due to the increase in Ca2+ influx via L‐type VDCCs.
Cellular Localization and Temporal Elevation of Tumor Necrosis Factor‐α, Interleukin‐1α, and Transforming Growth Factor‐β1 mRNA in Hippocampal Injury Response Induced by Trimethyltin
Authors: Bruccoleri Alessandra , Brown Hayes , Harry G. Jean
Published: July 2010

Abstract: In certain pathologic states, cytokine production may become spatially and temporally dysregulated, leading to their inappropriate production and potentially detrimental consequences. Tumor necrosis factor‐α (TNF‐α), interleukin (IL)‐1, IL‐6, and transforming growth factor‐β (TGF‐β) mediate a range of host responses affecting multiple cell types. To study the role of cytokines in the early stages of brain injury, we examined alterations in the 17‐day‐old mouse hippocampus during trimethyltin‐induced neurodegeneration characterized by neuronal necrosis, microglia activation in the dentate, and astrocyte reactivity throughout the hippocampus. By 24 h after dosing, elevations in mRNA levels for TNF‐α, IL‐1α, IL‐1β, and IL‐6 mRNA were seen. TGF‐β1 mRNA was elevated at 72 h. In situ hybridization showed that TNF‐α and IL‐1α were localized to the microglia, whereas TGF‐β1 was expressed predominantly in hippocampal pyramidal cells. Intercellular adhesion molecule‐1, EB‐22, Mac‐1, and glial fibrillary acidic protein mRNA levels were elevated within the first 3 days of exposure in the absence of increased inducible nitric oxide synthetase and interferon‐γ mRNA. These data suggest that pro‐inflammatory cytokines contribute to the progression and pattern of neuronal degeneration in the hippocampus.
Role of Carnitine Palmitoyltransferase I in the Control of Ketogenesis in Primary Cultures of Rat Astrocytes
Authors: Blázquez Cristina , Sánchez Cristina , Velasco Guillermo , Guzmán Manuel
Published: July 2010

Abstract: The role of carnitine palmitoyltransferase I (CPT‐I) in the control of ketogenesis was studied in primary cultures of rat astrocytes. Ketone bodies were the major product of [14C]palmitate oxidation by cultured astrocytes, whereas CO2 made a minor contribution to the total oxidation products. Using tetradecylglycidate as a specific, cell‐permeable inhibitor of CPT‐I, a flux control coefficient of 0.77 ± 0.07 was calculated for CPT‐I over the flux of [14C]palmitate to ketone bodies. CPT‐I from astrocytes was sensitive to malonyl‐CoA (IC50 = 3.4 ± 0.8 µM) and cross‐reacted on western blots with an antibody raised against liver CPT‐I. On the other hand, astrocytes expressed significant acetyl‐CoA carboxylase (ACC) activity, and consequently they contained considerable amounts of malonyl‐CoA. Western blot analysis of ACC isoforms showed that ACC in astrocytes—like in neurons, liver, and white adipose tissue—mostly comprised the 265‐kDa isoform, whereas the 280‐kDa isoform—which was highly expressed in skeletal muscle—showed much lower abundance. Forskolin was used as a tool to study the modulation of the ketogenic pathway in astrocytes. Thus, forskolin decreased in parallel ACC activity and intracellular malonyl‐CoA levels, whereas it stimulated CPT‐I activity and [14C]palmitate oxidation to both ketone bodies and CO2. Results show that in cultured astrocytes (a) CPT‐I exerts a very high degree of control over ketogenesis from palmitate, (b) the ACC/malonyl‐CoA/CPT‐I system is similar to that of liver, and (c) the ACC/malonyl‐CoA/CPT‐I system is subject to regulation by cyclic AMP.
Apolipoprotein E Attenuates β‐Amyloid‐Induced Astrocyte Activation
Authors: Hu Jingru , LaDu Mary Jo , Van Eldik Linda J.
Published: July 2010

Abstract: A common feature of Alzheimer's disease pathology is an abundance of activated glia, indicative of an inflammatory reaction in the brain. The relationship between glial activation and neurodegeneration is not known, although several cytokines and inflammatory mediators produced by activated glia have the potential to initiate or exacerbate the progression of neuropathology. As β‐amyloid (Aβ) is one of several stimuli that can activate glia, it is important to determine how Aβ‐induced glial activation is influenced by other proteins present in the plaque, such as apolipoprotein E (apoE). We examined the effect of native preparations of apoE on activation of rat cortical astrocyte cultures by Aβ1–42. The apoE source was conditioned medium from human embryonic kidney 293 cells stably transfected with human apoE3 or apoE4 cDNA. By morphological criteria, apoE inhibited Aβ‐induced astrocyte activation in three experimental paradigms: apoE pretreatment blocked subsequent Aβ‐induced activation, Aβ aged in the presence of apoE did not activate astrocytes, and apoE addition to activated astrocytes transiently reversed the activated phenotype. No apoE isoform selectivity was observed. The effect of apoE appears to be specific to Aβ, as apoE did not attenuate cyclic AMP‐induced astrocyte activation. These data suggest that apoE may modulate the ability of Aβ to induce inflammatory responses in the brain.
Aspirin and Salicylate Protect Against MPTP‐Induced Dopamine Depletion in Mice
Authors: Aubin Nadine , Curet Olivier , Deffois Annie , Carter Chris
Published: July 2010

Abstract: The neurotoxic effects of the dopamine‐selective neurotoxin MPTP (15 mg/kg, s.c.), in mice, were totally prevented by systemic administration of salicylate (ED50 = 40 mg/kg, i.p.), aspirin (ED50 = 60 mg/kg, i.p.), or the soluble lysine salt of aspirin, Aspegic (ED50 = 80 mg/kg, i.p.). The protective effects of aspirin are unlikely to be related to cyclooxygenase inhibition as paracetamol (100 mg/kg, i.p.), diclofenac (100 mg/kg, i.p.), ibuprofen (20 mg/kg, i.p.) and indomethacin (100 mg/kg, i.p.) were ineffective. Dexamethasone (3–30 mg/kg, i.p.), which, like aspirin and salicylate, has been reported to inhibit the transcription factor NF‐κβ, was also ineffective. Aspirin or salicylate (100 µM) had no effect on dopamine uptake into striatal synaptosomes or on monoamine oxidase B activity. The neuroprotective effects of salicylate derivatives could perhaps be related to hydroxyl radical scavenging. This was suggested by the fact that hydroxylated metabolites of salicylate (2,3‐ and 2,5‐dihydrobenzoic acid) were recovered in brain tissue following the combined administration of MPTP and aspirin to a greater extent than following aspirin alone. The surprising neuroprotective effects of aspirin in an animal model of Parkinson's disease warrant further clinical investigation.
Role of Serine‐19 Phosphorylation in Regulating Tyrosine Hydroxylase Studied with Site‐ and Phosphospecific Antibodies and Site‐Directed Mutagenesis
Authors: Haycock J. W. , Lew J. Y. , Garcia‐Espana A. , Lee K. Y. , Harada K. , Meller E. , Goldstein M.
Published: July 2010

Abstract: The effects of depolarization by elevated potassium concentrations were studied in PC12 cells and in stably transfected AtT‐20 cells expressing wild‐type or [Leu19]‐recombinant tyrosine hydroxylase (rTH). Changes in the phosphorylation states of Ser19 and Ser40 in tyrosine hydroxylase (TH) were determined immunochemically using antibodies specific for the phosphorylated state of each site and compared with changes in TH activity in PC12 cell lysates and with changes in l‐DOPA biosynthesis rates in intact AtT‐20 cells. Treatment of either PC12 cells or AtT‐20 cells expressing wild‐type rTH with elevated potassium produced a transient increase in the phosphorylation state of Ser19 (up to 0.7 mol of phosphate/mol of subunit) in concert with a more gradual and sustained increase in Ser40 phosphorylation. Elevated potassium treatment also increased TH activity in PC12 cell lysates, but these increases paralleled the temporal course of Ser40, as opposed to Ser19, phosphorylation. Similarly, increases in DOPA accumulation produced by elevated potassium in AtT‐20 cells expressing wild‐type rTH paralleled the increases in the phosphorylation state of Ser40 but not Ser19. Moreover, elevated potassium produced comparable increases in DOPA accumulation in AtT‐20 cells expressing rTH in which Ser19 phosphorylation had been eliminated (by substitution of Leu for Ser19). Thus, depolarization‐induced increases in the stoichiometry of Ser19 phosphorylation do not appear to influence directly the activity of TH in situ.
Ca2+‐ and Mg2+‐Modulated Lipolysis in Neonatal Rat Brain Slices Observed by One‐ and Two‐Dimensional NMR
Authors: Gasparovic Charles , Berghmans Kirsten
Published: July 2010

Abstract: Superfused cortical brain slices from neonatal rats demonstrated large increases in levels of NMR‐detectable lipids after sample preparation and perfusion with standard artificial CSF. These increases were reduced by an average of 58% by perfusion with buffer with low (no added) Ca2+ or by perfusion in Ca2+‐free buffer. Perfusion with buffer with elevated MgSO4 (10 mmol/L) reduced the lipid changes by 47%. A reduction of 88% was observed in samples perfused in buffer with both low Ca2+ and high Mg2+, suggesting a role for Mg2+ in reducing lipolysis distinct from its known ability to block Ca2+ influx.
Up‐Regulation of Blood‐Brain Barrier Short‐Form Leptin Receptor Gene Products in Rats Fed a High Fat Diet
Authors: Boado Ruben J. , Golden Pamela L. , Levin Nancy , Pardridge William M.
Published: July 2010

Abstract: Leptin is a 16‐kDa protein synthesized in adipose tissue that produces a satiety effect in the CNS. Leptin may gain access to the brain via receptor‐mediated transport through the blood‐brain barrier (BBB), and the BBB leptin receptor (OBR) may regulate the availability of circulating leptin to brain cells. The aim of the present study was twofold: first, to identify the OBR isoform expressed at the BBB, i.e., short, or “a,” and long, or “b,” form; and second, to compare the abundance of the BBB OBR mRNA and protein between control and high fat‐fed rats. RT‐PCR with isoform‐specific primers showed that OBRa is the most abundant isoform at the BBB. BBB OBRa transcript content was markedly increased in high fat‐fed rats compared with controls (11‐fold), and no changes were observed in the expression of the internal standard control actin. The high fat feeding induction of OBR mRNA was correlated with an increase in the immunoreactive BBB OBR determined by immunocytochemistry using an all‐isoform reactive antibody in high fat‐fed obese rats. This investigation demonstrates (a) the OBRa is the principal leptin receptor expressed at the BBB and (b) this BBB OBR isoform is up‐regulated by a high fat diet.
Progesterone Stimulates the Activity of the Promoters of Peripheral Myelin Protein‐22 and Protein Zero Genes in Schwann Cells
Authors: Désarnaud Frank , Do Thi Anh N. , Brown Adrienne M. , Lemke Greg , Suter Ueli , Baulieu Etienne‐Emile , Schumacher Michael
Published: July 2010

Abstract: To understand better the mechanisms by which progesterone (PROG) promotes myelination in the PNS, cultured rat Schwann cells were transiently transfected with reporter constructs in which luciferase expression was controlled by the promoter region of either the peripheral myelin protein‐22 (PMP22) or the protein zero (P0) genes. PROG stimulated the P0 promoter and promoter 1, but not promoter 2, of PMP22. The effect of PROG was specific, as estradiol and testosterone only weakly activated promoters. Dose‐response curves for stimulation of both promoter constructs by PROG were biphasic. RU486, a PROG antagonist, did not abolish the effect of PROG, but stimulated promoter activities by itself. In the human carcinoma cell line T47D expressing high levels of PROG receptor, PROG did not stimulate the P0 and PMP22 promoters, whereas the promoter region of the mouse mammary tumor virus was fully activated. Thus, the activation by PROG of promoter activity of two peripheral myelin protein genes is Schwann‐cell specific.
Acute Effects of Ethanol on Pharmacologically Isolated Kainate Receptors in Cerebellar Granule Neurons: Comparison with NMDA and AMPA Receptors
Authors: Valenzuela C. Fernando , Bhave Sanjiv , Hoffman Paula , Harris R. Adron
Published: July 2010

Abstract: Comparisons of acute ethanol's effects on individual members of the three major families of ionotropic glutamate receptors (kainate, AMPA, and NMDA) have been performed only with recombinant receptors. However, no study has compared the acute effects of ethanol on individual members of each one of these receptor families in the same neuron. We accomplished this task by using cultured cerebellar granule neurons and LY303070 (GYKI‐53784), a noncompetitive and selective AMPA receptor antagonist. Ethanol concentrations of 25, 50, 75, and 100 mM decreased the amplitude of pharmacologically isolated kainate‐activated currents by 3 ± 1, 9 ± 2, 14 ± 2, and 22 ± 3% (n = 8), respectively. The magnitude of the ethanol‐induced inhibition of nonselective kainate‐activated currents, i.e., in the absence of LY303070, and currents activated by submaximal AMPA concentrations was not significantly different from that obtained with isolated kainate currents. However, the magnitude of the ethanol‐induced inhibition of NMDA receptor‐activated currents was about twofold greater than that of kainate and/or AMPA receptors.
Paired‐Like Homeodomain Proteins, Phox2a and Phox2b, Are Responsible for Noradrenergic Cell‐Specific Transcription of the Dopamine β‐Hydroxylase Gene
Authors: Yang Chunying , Kim Hee‐Sun , Seo Hyemyung , Kim Chun‐Hyung , Brunet Jean‐Francois , Kim Kwang‐Soo
Published: July 2010

Abstract: Recently, a murine paired‐like homeobox gene, Phox2a, has been identified whose product is critical for the development of several major noradrenergic neuron populations, including the locus coeruleus. In noradrenergic neurons, dopamine β‐hydroxylase (DBH) is a hallmark protein and catalyzes the conversion of dopamine to noradrenaline. Our previous studies have shown that a composite promoter (domain IV), residing at −185 to −150 bp upstream of the transcription start site, is critical for DBH transcription and is comprised of multiple cis‐acting elements, including a cyclic AMP response element, a YY1 binding site, and two core motifs of the homeodomain (HD)‐binding site. Here, we show that the HD‐binding site residing within domain IV is a noradrenergic‐specific cis‐acting element. In contrast, the cyclic AMP response element is active in all cell lines tested. We provide evidence that Phox2a is expressed only in DBH‐positive cell lines and interacts with the HD‐binding site. Forced expression of Phox2a robustly activates DBH promoter activity in DBH‐negative cell lines (>10‐fold), but increased it only marginally (<50%) in DBH‐positive cell lines. Furthermore, another protein factor with an identical HD, Phox2b, also activates DBH transcription with an efficiency comparable to that of Phox2a. In contrast, neither Phox2a nor Phox2b was able to transactivate tyrosine hydroxylase transcription, indicating that these transcription factors differentially activate catecholamine‐synthesizing gene transcription. Together with the Phox2a knockout experiment, the studies described here make Phox2a and Phox2b the first strong candidate transcription factors for determining a neurotransmitter phenotype in vertebrates.
Interleukin‐6‐Mediated Autocrine Growth Promotion in Human Glioblastoma Multiforme Cell Line U87MG
Authors: Goswami Sumanta , Gupta Achla , Sharma Shail K.
Published: July 2010

Abstract: Human glioblastoma multiforme cell lines, brain tumor biopsy tissue, and normal human fetal brain synthesize interleukin (IL)‐6 and IL‐6 receptor (IL‐6R). Neither of these is expressed in human neurons or neuroblastoma cell lines in culture. Astrocytes from fetal brain grown in culture retain the ability to synthesize IL‐6 but do not express IL‐6R as inferred from RT‐PCR and Southern blot studies. Coexpression of IL‐6 and IL‐6R in the glioblastoma cell line U87MG is confirmed by immunofluorescence staining. Both specific monoclonal antibodies against IL‐6 and IL‐6R and antisense oligonucleotide to IL‐6 mRNA inhibit the growth of U87MG cells in culture, suggesting the existence of a functional autocrine growth loop. Anti‐IL‐6 antibodies also inhibit the growth of glioblastoma cell lines U373 and U118. The expression of IL‐6 by human fetal astrocytes in culture is highly suggestive of its role as an oncofetal protein responsible for rapid proliferation of fetal and tumor cells but not cells of adult brain.
Regulation of Sensory Neuron Precursor Proliferation by Cyclic GMP‐Dependent Protein Kinase
Authors: Firestein Bonnie L. , Bredt David S.
Published: July 2010

Abstract: Cyclic GMP (cGMP) is a molecular messenger involved in diverse cellular processes. Recently, cGMP‐dependent protein kinase (cGK) type II was determined to be a regulator of endochondral ossification and bone growth, identifying a role for cGMP in the regulation of cellular proliferation. Here, we demonstrate the presence of cGK type I (cGKI) in cells of the developing trigeminal ganglia. cGKI occurs in some proliferating precursors as evidenced by double labeling with an antibody to cGKI and 5‐bromo‐2′‐deoxyuridine(BrdU) incorporation. Inhibition of cGKI with KT5823 or Rp‐8‐(4‐chlorophenylthio)‐guanosine‐3′,5′‐cyclic monophosphorothioate (Rp‐8‐pCPT‐cGMPS) in chick embryos results in a 30–40% decrease in trigeminal ganglia cell number, and this effect is independent of nitric oxide synthase (NOS). In addition, inhibition of cGKI with Rp‐8‐pCPT‐cGMPS results in a 60% decrease in BrdU incorporation in the trigeminal ganglia of embryonic day 5 chicks. We find that PC12 cells expressing cGKI proliferate more rapidly and incorporate more BrdU than do control cells. The cGKI inhibitor Rp‐8‐pCPT‐cGMPS decreases proliferation and BrdU incorporation in transfected PC12 cells but has no effect on control cells. The PC12 cells do not express NOS, indicating that this effect is also independent of NOS. Thus, cGKI regulates the proliferation of sensory neurons as a result of activation of a NOS‐independent pathway, representing a novel pathway by which the number of sensory neurons is regulated.
Cell Type‐Specific Regulation of CREB Gene Expression: Mutational Analysis of CREB Promoter Activity
Authors: Coven Emily , Ni Yan , Widnell Katherine L. , Chen Jingshan , Walker William H. , Habener Joel F. , Nestler Eric J.
Published: July 2010

Abstract: Previous studies have shown that activation of the cyclic AMP (cAMP) pathway down‐regulates CREB expression in CATH.a cells, an effect that appears to be mediated via inhibition of CREB gene transcription. In the current study, we compared this effect in CATH.a cells with regulation of CREB expression in another cell line, C6 glioma cells. In contrast to the findings in CATH.a cells, activation of the cAMP pathway up‐regulates CREB expression in C6 glioma cells. To determine whether these opposite effects can be explained by regulation of CREB promoter activity, chloramphenicol acetyltransferase (CAT) assays were performed in CATH.a and C6 glioma cells that were transiently transfected with a CREB promoter‐CAT fusion plasmid. Activation of the cAMP pathway decreased levels of CAT activity in transfected CATH.a cells but increased CAT activity in transfected C6 glioma cells. We next investigated the effect of mutations in the CREB promoter on such regulation in these two cell lines. Mutations of single CRE or Sp1 binding sites in the CREB promoter reduced basal levels of CAT activity but did not significantly attenuate regulation of the promoter in CATH.a or C6 glioma cells. However, mutation or deletion of two CRE sites in the CREB promoter completely abolished up‐regulation of CAT activity in the C6 glioma cells and abolished basal levels of CAT activity in CATH.a cells. CREB promoter activity was also studied in cultured SHSY5Y cells and in primary cultures of striatal neurons as further comparisons. Activation of the cAMP pathway was found to increase CAT activity in both cell types. In the striatal cultures, this effect was obliterated by mutation or deletion of either of the two CREs in the promoter. These findings demonstrate cell type‐specific effects of the cAMP pathway on CREB expression, which appear to be mediated via differential regulation of the CREB promoter.
Ornithine Decarboxylase Activity During Development of Cerebellar Granule Neurons
Authors: Sparapani Mauro , Virgili Marco , Bardi Giuseppe , Tregnago Manuela , Monti Barbara , Bentivogli Monia , Contestabile Antonio
Published: July 2010

Abstract: Ornithine decarboxylase (ODC), the key enzyme for polyamine biosynthesis, dramatically decreases in activity during normal cerebellar development, in parallel with the progressive differentiation of granule neurons. We have studied whether a similar pattern is displayed by cerebellar granule neurons during survival and differentiation in culture. We report that when granule cells were kept in vitro under trophic conditions (high K+ concentration), ODC activity progressively decreased in parallel with neuronal differentiation. Under nontrophic conditions (cultures kept in low K+ concentration), the enzymatic activity dropped quickly in parallel with an increased apoptotic elimination of cells. Cultures kept in high K+ but chronically exposed to 10 mM lithium showed both an increased rate of apoptotic cell death at 2 and 4 days in vitro and a quicker drop of ODC activity and immunocytochemical staining. A short chronic treatment of rat pups with lithium also resulted in transient decrease of cerebellar ODC activity and increased programmed cell death, as revealed by in situ detection of apoptotic granule neurons. The present data indicate that a sustained ODC activity is associated with the phase of survival and differentiation of granule neurons and that, conversely, conditions that favor their apoptotic elimination are accompanied by a down‐regulation of the enzymatic activity.
Characterization of the Phosphoinositide‐Linked Dopamine Receptor in a Mouse Hippocampal‐Neuroblastoma Hybrid Cell Line
Authors: Jin Li‐Qing , Cai Guoping , Wang Hoau‐Yan , Smith Candyce , Friedman Eitan
Published: July 2010

Abstract: Previous studies have established that dopamine (DA) can stimulate phosphoinositide (PI) metabolism in the CNS and in the periphery. The present study summarizes our attempt to find a cell line that expresses this dopaminergic system. We describe that the stable clonal HN33.11 cell line, established by fusion of mouse hippocampal cells with neuroblastoma cells (N18TG2) that originate from A/J mouse, natively expresses the D1 DA receptor system that couples to PI hydrolysis. In this cell line, 500 µM DA or SKF38393 produced 43 and 75% increases in inositol phosphate (IP) accumulations, respectively. In contrast, noradrenaline or 5‐hydroxytryptamine did not affect IP accumulations. The formation of IP that was stimulated by DA or SKF38393 was selectively blocked by the D1 DA receptor antagonist SCH23390 with IC50 values of 13 and 16 µM. This response was not mediated by the D1A DA receptor and was cyclic AMP‐independent, as HN33.11 cells did not express this receptor, and DA or SKF38393 was unable to stimulate the formation of cyclic AMP. In Ca2+‐free/100 µM EGTA medium, basal IP level was reduced by 31.5%, but SKF38393‐stimulated PI hydrolysis was not affected. SKF38393‐stimulated IP accumulation was also not affected by pertussis toxin (PTX) treatment (200 ng/ml), suggesting that this dopaminergic response is mediated by PTX‐insensitive G proteins. Co‐immunoprecipitation studies indicated that in membranes of HN33.11 cells, D1‐like binding sites are coupled to Gαq protein. Blockade of SKF38393‐induced PI hydrolysis with antiserum against phospholipase C (PLC) isozymes, performed in permeabilized cells, as well as co‐immunoprecipitation studies implicate PLCβ3 and PLCβ4 in this dopaminergically mediated PI hydrolysis cascade. The results indicate that HN33.11 cells express a D1‐like DA receptor that couples to PLCβ3/4 via Gαq protein. These cells may therefore be a useful model system for investigating this receptor system.