bluuebunny/crossref_metadata_2025 huggingface dataset:
Kainate‐Induced Apoptosis in Cultured Murine Cerebellar Granule Cells Elevates Expression of the Cell Cycle Gene Cyclin D1
Authors: Giardina Sarah F. , Cheung Nam S. , Reid Michelle T. , Beart Philip M.
Published: July 2010

Abstract: Recent evidence suggests that neuronal apoptosis is the consequence of an inappropriate reentry into the cell cycle. Expression of the cell cycle gene cyclin D1, a G1‐phase cell cycle regulator, was examined in primary cultures of murine cerebellar granule cells (CGCs) during kainate (KA)‐mediated apoptosis. Using cultures of CGCs, we found that a 24‐h exposure to KA (1–3,000 µM) induced a concentration‐dependent cell death with neurons exhibiting characteristic apoptotic morphology and extensive labeling using the terminal transferase‐mediated nick end‐DNA labeling (TUNEL) method. KA induced a time‐ and concentration‐dependent increase in expression of cyclin D1 as determined by immunocytochemistry and western blot analysis. KA‐induced apoptosis and cyclin D1 expression exhibited a similar concentration dependence and were significantly attenuated by the non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (50 µM), indicating a KA receptor‐mediated effect. Here we present evidence for the first time that KA‐induced apoptosis in cultured CGCs involves the induction of cyclin D1, suggesting its involvement in excitotoxic receptor‐mediated apoptosis.
Identification and Characterization of Potential cis‐Regulatory Elements Governing Transcriptional Activation of the Rat Tyrosine Hydroxylase Gene
Authors: Yang Chunying , Kim Hee‐Sun , Seo Hyemyung , Kim Kwang‐Soo
Published: July 2010

Abstract: Tyrosine hydroxylase (TH) catalyzes the conversion of l‐tyrosine to 3,4‐dihydroxy‐l‐phenylalanine, which is the first and rate‐limiting step in catecholamine biosynthesis. We have previously shown that the cyclic AMP response element (CRE), an essential promoter element for both basal and cyclic AMP‐inducible TH transcription, activates the promoter activity in a distance‐dependent manner. To identify further cis‐regulatory elements controlling TH gene expression, we analyzed the potential regulatory sequences by several approaches. First, using transient transfection assays, we examined the cell‐specific promoter activities of TH‐reporter gene constructs and a dopamine β‐hydroxylase (DBH)‐reporter construct containing the 5′ upstream sequences of the rat TH and human DBH genes, respectively, that had been shown to direct tissue‐specific reporter expression in transgenic mice experiments. Second, DNase I footprinting analysis of the 503‐bp proximal area of the rat TH gene identified seven footprinted regions that encompass the putative cis‐regulatory motifs, including the CRE (domain I), Sp1 (domain III), Octamer (domain IV), AP1 (domain V), AP2 (domain VI), and two potentially novel sequence motifs (domains II and VII). Footprinting patterns at these sites by nuclear proteins from TH‐positive and ‐negative cell lines appeared to be similar. Third, site‐directed mutagenesis demonstrates that domain III, but not domain II, critically contributes to the TH promoter activity. Furthermore, electrophoretic mobility shift, competition, and supershift assays demonstrate that domain III is an authentic Sp1 site and that the transcription factor Sp1 interacts with it. This and previous results suggest that the CRE and Sp1 site may synergistically activate TH transcription in a promoter context‐dependent manner.
Activation of Excitatory Amino Acid Receptors in the Rat Hippocampal Slice Increases Intracellular Cl− and Cell Volume
Authors: Inglefield Jon R. , Schwartz‐Bloom Rochelle D.
Published: July 2010

Abstract: The effects of glutamatergic excitotoxins on intracellular Cl− were investigated in the CA1 pyramidal cell layer of the hippocampal slice. Hippocampal slices from rats (14–19 days old) were loaded with 6‐methoxy‐N‐ethylquinolinium chloride (MEQ), a Cl−‐sensitive fluorescent probe with a fluorescence intensity that correlates inversely with intracellular [Cl−]. Slices were exposed for at least 10 min at 26–28°C to N‐methyl‐d‐aspartate (NMDA; 100 µM) or α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA; 50 µM). A UV laser scanning confocal microscope was used to measure changes in MEQ fluorescence within area CA1 pyramidal cell soma. Both glutamate receptor agonists produced a rapid decrease in MEQ fluorescence that persisted after washout following a 10‐min exposure. The effects of NMDA and AMPA were prevented by the competitive antagonists 2‐amino‐5‐phosphonopentanoic acid and 6,7‐dinitroquinoxaline‐2,3‐dione, respectively. Neither tetrodotoxin nor picrotoxin prevented the effect of NMDA or AMPA, indicating the lack of involvement of presynaptic mechanisms. The effects of NMDA and AMPA on MEQ fluorescence were dependent on the levels of extracellular Cl−, but only NMDA responses were dependent on the levels of extracellular Na+. Removal of Ca2+ from the superfusion medium did not alter the effects of NMDA or AMPA on MEQ fluorescence. In addition, neither the Ca2+ ionophore ionomycin nor the L‐type voltage‐gated Ca2+ channel agonist (Bay K 8644) decreased MEQ fluorescence. The effects of NMDA and AMPA on cell (somal) volume were also assessed with the fluorescent probe calcein acetoxymethyl ester. Both NMDA and AMPA decreased calcein fluorescence (indicating an increased cell volume), but this was preceded by the decrease in MEQ fluorescence (equivalent to an intracellular accumulation of ∼20 mM Cl−). Thus, excitotoxins may cause Cl− influx via an anion channel other than the GABAA receptor and/or reduce Cl− efflux mechanisms to produce cell swelling. Such anionic shifts may promote neuronal excitability and cell death following an excitotoxic insult to the hippocampal slice.
Differential Effects of Two Protein Kinase C Inhibitors, Calphostin C and Gö6976, on Pineal Cyclic Nucleotide Accumulation
Authors: Ogiwara T. , Negishi T. , Chik C. L. , Ho A. K.
Published: July 2010

Abstract: In rat pinealocytes, protein kinase C (PKC) is involved in the α1‐adrenergic‐mediated potentiation of β‐adrenergic‐stimulated cyclic nucleotide responses; however, the specific PKC isozyme(s) involved in the potentiation mechanism remain unknown. In the present study, we compared the effects of two PKC inhibitors, calphostin C, a specific inhibitor of PKC, and Gö6976, a selective inhibitor of PKCα and PKCβ1, on the adrenergic‐stimulated cyclic nucleotide accumulation in rat pinealocytes. Surprisingly, Gö6976 was found to have an enhancing effect on basal cyclic GMP and isoproterenol‐stimulated cyclic AMP and cyclic GMP accumulation, an effect not shared by calphostin C. Gö6976 also increased the norepinephrine‐ and ionomycin‐induced potentiation of isoproterenol‐stimulated cyclic AMP and cyclic GMP accumulation, whereas the effect of calphostin C was inhibitory. The enhancing effect of Gö6976 was abolished in the presence of isobutylmethylxanthine or zaprinast, but not rolipram, suggesting that this effect of Gö6976 may be mediated through type V or the retinal type of phosphodiesterase. Based on these observations, we propose that some of the PKC isozyme(s) inhibited by calphostin C are involved in the potentiation of β‐adrenergic‐stimulated cyclic nucleotide responses and that they act by enhancing synthesis. However, PKC isozymes inhibited by Gö6976 appear to be basally active and tonically inhibit cyclic nucleotide accumulation through their stimulatory action on phosphodiesterase.
Long‐Term Effects of Portacaval Anastomosis on the 5‐Hydroxytryptamine, Histamine, and Catecholamine Neurotransmitter Systems in Rat Brain
Authors: Lozeva V. , MacDonald E. , Belcheva A. , Hippeläinen M. , Kosunen H. , Tuomisto L.
Published: July 2010

Abstract: Portacaval anastomosis (PCA) in the rat is used as a model for portal systemic encephalopathy. Changes in the serotonergic, histaminergic, and catecholaminergic neurotransmitter systems are often found shortly after PCA. We have examined the long‐term effects of PCA on the aminergic systems in brains of male Wistar rats, which 8 months previously had been subjected to PCA. Precursors, amines, and metabolites were assayed by HPLC. Eight months after PCA, the catecholamine levels were unchanged in all brain regions. In contrast, tryptophan was evenly increased throughout the brain. The accumulation of 5‐hydroxytryptophan after decarboxylase inhibition (NSD‐1015; 100 mg/kg i.p.) and the endogenous levels of 5‐hydroxyindoleacetic acid were significantly higher in PCA rats, particularly in the hypothalamus and midbrain, whereas 5‐hydroxytryptamine concentrations were unchanged. Histamine levels were elevated throughout the brain with the greatest increase found in the hypothalamus and in the striatum. tele‐Methylhistamine levels were significantly elevated in cortex and hypothalamus. We conclude that 8 months after PCA, catecholaminergic systems had reestablished their homeostasis, whereas serotonergic and histaminergic systems still show profound disturbances in their function. With histamine, this is reflected as an increase in the amounts of both transmitter and metabolite; serotonergic neurons respond by increasing only the level of the metabolite.
Regulation of Serotonin2A Receptor Expression by an Antisense Oligodeoxynucleotide
Authors: Scalzitti J. M. , Berg K. A. , Kratowicz S. A. , Hensler J. G.
Published: July 2010

Abstract: The regulation of 5‐HT2A receptor expression by an antisense oligodeoxynucleotide, complementary to the coding region of rat 5‐HT2A receptor mRNA, was examined in a cortically derived cell line and in rat brain. Treatment of A1A1 variant cells, which express the 5‐HT2A receptor coupled to the stimulation of phosphatidylinositol (PI) hydrolysis, with antisense oligodeoxynucleotide decreased the maximal stimulation of PI hydrolysis by the partial agonist quipazine and the number of 5‐HT2A receptor sites as measured by the binding of 2‐[125I]‐iodolysergic acid diethylamide. Treatment of cells with random, sense, or mismatch oligodeoxynucleotide did not alter the stimulation of PI hydrolysis by quipazine or 5‐HT2A receptor number. Intracerebroventricular infusion of antisense, but not mismatch, oligodeoxynucleotide for 8 days resulted in a significant increase in cortical 5‐HT2A receptor density and an increase in headshake behavior induced by the 5‐HT2 receptor agonist 1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane. The density of cortical 5‐HT2A receptors was not altered by administration of antisense oligodeoxynucleotide for 1, 2, or 4 days. We hypothesize that in brain this antisense oligodeoxynucleotide relieved some form of translational suppression, resulting in an increase in 5‐HT2A receptor expression.
Hypoxia Enhances [3H]Noradrenaline Release Evoked by Nicotinic Receptor Activation from the Human Neuroblastoma SH‐SY5Y
Authors: Wade J. A. , Vaughan P. F. T. , Peers C.
Published: July 2010

Abstract: We have used the human sympathetic neuronal line SH‐SY5Y to investigate the effects of hypoxia on noradrenaline (NA) release evoked by either raised [K+]o (100 mM) or the nicotinic acetylcholine receptor (nAChR) agonist dimethylphenylpiperazinium iodide (DMPP). NA release was monitored by loading cells with [3H]NA and collecting effluent fractions from perfused cells kept in a sealed perifusion chamber. Cells were challenged twice with either stimulus and release was expressed as that evoked by the second challenge as a fraction of that evoked by the first. K+‐evoked release was unaffected by hypoxia (Po2≅ 30–38 mm Hg), but release evoked by DMPP was significantly increased. For both stimuli, replacement of Ca2+o with 1 mM EGTA abolished NA release. K+‐evoked release was also dramatically reduced in the presence of 200 µM Cd2+ to block voltage‐gated Ca2+ channels, but DMPP‐evoked release was less affected. In hypoxia, DMPP‐evoked Cd2+‐resistant NA release was dramatically increased. Our findings indicate that hypoxia increases NA release evoked from SH‐SY5Y cells in response to nAChR activation by increasing Ca2+ influx through the nAChR pore, or by activating an unidentified Cd2+‐resistant Ca2+‐influx pathway. As acetylcholine is the endogenous transmitter at sympathetic ganglia, these findings may have important implications for sympathetic activity under hypoxic conditions.
Calcium Modulation of Activation and Desensitization of Nicotinic Receptors from Mouse Brain
Authors: Booker T. K. , Smith K. W. , Dodrill C. , Collins A. C.
Published: July 2010

Abstract: The effects of extracellular calcium on functional properties of nicotinic receptors from mouse thalamus were investigated. Previous studies have reported that calcium modulates the function of several neuronal nicotinic receptors. A 86Rb+ ion efflux assay was developed to measure nicotinic receptor function from brain tissue, and data indicate that α4β2 receptors may mediate this response. Using the 86Rb+ efflux assay, calcium effects on receptor activation, desensitization induced by high, activating and low, subactivating concentrations of agonist, and recovery from desensitization were examined. Effects of calcium on the kinetics of ligand binding were also investigated. Calcium modulated receptor activation by increasing the maximal response to nicotine in a concentration‐dependent manner, without affecting the EC50 of nicotine. Barium, but not magnesium, mimicked the effects of calcium on receptor activation. The increase in receptor activation could not be explained by changes in the ratio of activatable to desensitized receptors as assessed by the kinetics of ligand binding. Desensitization following activation was unaffected by calcium. Calcium, barium, and magnesium, however, increased the potency of nicotine for desensitization induced by exposure to low, subactivating concentrations of nicotine. Recovery from desensitization was not modulated by calcium. These data suggest that calcium modulates various functional aspects of nicotinic receptors from mouse brain and may do so via different mechanisms.
Quantification of the GABA Shunt and the Importance of the GABA Shunt Versus the 2‐Oxoglutarate Dehydrogenase Pathway in GABAergic Neurons
Authors: Hassel Bjørnar , Johannessen Cecilie U. , Sonnewald Ursula , Fonnum Frode
Published: July 2010

Abstract: We investigated the activity of the cerebral GABA shunt relative to the overall cerebral tricarboxylic acid (TCA) cycle and the importance of the GABA shunt versus 2‐oxoglutarate dehydrogenase for the conversion of 2‐oxoglutarate into succinate in GABAergic neurons. Awake mice were dosed with [1‐13C]glucose, and brain extracts were analyzed by 13C NMR spectroscopy. The percent enrichments of GABA C‐2 and glutamate C‐4 were the same: 5.0 ± 1.6 and 5.1 ± 0.2%, respectively (mean ± SD). This, together with previous data, indicates that the flux through the GABA shunt relative to the overall cerebral TCA cycle flux equals the GABA/glutamate pool size ratio, which in the mouse is 17%. It has previously been shown that under the experimental conditions used in this study, the 13C labeling of aspartate from [1‐13C]glucose specifically reflects the metabolic activity of GABAergic neurons. In the present study, the reduction in the formation of [13C]aspartate during inhibition of the GABA shunt by γ‐vinyl‐GABA indicated that not more than half the flux from 2‐oxoglutarate to succinate in GABAergic neurons goes via the GABA shunt. Therefore, because fluxes through the GABA shunt and 2‐oxoglutarate dehydrogenase in GABAergic neurons are approximately the same, the TCA cycle activity of GABAergic neurons could account for one‐third of the overall cerebral TCA cycle activity in the mouse. Treatment with γ‐vinyl‐GABA, which increased GABA levels dramatically, caused changes in the 13C labeling of glutamate and glutamine, which indicated a reduction in the transfer of glutamate from neurons to glia, implying reduced glutamatergic neurotransmission. In the most severely affected animals these alterations were associated with convulsions.
Stimulation of Neuropeptide Y Overflow in the Rat Paraventricular Nucleus by Corticotropin‐Releasing Factor
Authors: Morris M. J. , Pavia J. M.
Published: July 2010

Abstract: Neuropeptide Y (NPY) and corticotropin‐releasing factor (CRF) are present at high concentrations in the hypothalamus where they mediate important endocrine and autonomic functions. Morphological and physiological studies have suggested an interaction between these peptides, and opposing actions of CRF and NPY have been reported on feeding and other behaviors. This study investigated the effect of CRF on NPY release in vivo, measured by push‐pull techniques, in the anesthetized rat. Push‐pull probes implanted into the paraventricular nucleus of the hypothalamus (PVN) were perfused with modified Ringer solution containing bovine serum albumin at 15 µl/min, and the perfusate was lyophilized prior to NPY radioimmunoassay. NPY overflow from the rat PVN was increased threefold by perfusion of a depolarizing concentration of potassium (50 mmol/L KCI). When CRF was administered into the PVN via the push‐pull cannula at 1 or 5 µg/ml, dose‐dependent increases in NPY overflow of two‐ and fivefold were observed (p < 0.05). These increases were abolished by prior intracerebroventricular (i.c.v.) administration of the CRF antagonist [d‐Phe12,Nle21,38,CαMeLeu32]CRF (12–41) at 1 or 5 µg/µl, respectively. NPY overflow returned promptly to resting levels following CRF administration. In contrast, when CRF was administered by i.c.v. bolus at a similar total dose (2 µg), no significant effect on NPY overflow was observed. These data provide in vivo evidence for an interaction between CRF and NPY at the level of the PVN.
Presenilin 1 Mutations Linked to Familial Alzheimer's Disease Increase the Intracellular Levels of Amyloid β‐Protein 1–42 and Its N‐Terminally Truncated Variant(s) Which Are Generated at Distinct Sites
Authors: Sudoh Shinji , Kawamura Yuuki , Sato Shinji , Wang Rong , Saido Takaomi C. , Oyama Fumitaka , Sakaki Yoshiyuki , Komano Hiroto , Yanagisawa Katsuhiko
Published: July 2010

Abstract: Mutations in the presenilin genes PS1 and PS2 cause the most common form of early‐onset familial Alzheimer's disease. The influence of PS1 mutations on the generation of endogenous intracellular amyloid β‐protein (Aβ) species was assessed using a highly sensitive immunoblotting technique with inducible mouse neuro‐blastoma (Neuro 2a) cell lines expressing the human wild‐type (wt) or mutated PS1 (M146L or Δexon 10). The induction of mutated PS1 increased the intracellular levels of two distinct Aβ species ending at residue 42 that were likely to be Aβ1–42 and its N‐terminally truncated variant(s) Aβx‐42. The induction of mutated PS1 resulted in a higher level of intracellular Aβ1–42 than of intracellular Aβx‐42, whereas extracellular levels of Aβ1–42 and Aβx‐42 were increased proportionally. In addition, the intracellular generation of these Aβ42 species in wt and mutated PS1‐induced cells was completely blocked by brefeldin A, whereas it exhibited differential sensitivities to monensin: the increased accumulation of intracellular Aβx‐42 versus inhibition of intracellular Aβ1–42 generation. These data strongly suggest that Aβx‐42 is generated in a proximal Golgi, whereas Aβ1–42 is generated in a distal Golgi and/or a post‐Golgi compartment. Thus, it appears that PS1 mutations enhance the degree of 42‐specific γ‐secretase cleavage that occurs in the normal β‐amyloid precursor protein processing pathway (a) in the endoplasmic reticulum or the early Golgi apparatus prior to β‐secretase cleavage or (b) in the distinct sites where Aβx‐42 and Aβ1–42 are generated.
7Li Nuclear Magnetic Resonance Study for the Determination of Li+ Properties in Neuroblastoma SH‐SY5Y Cells
Authors: Nikolakopoulos Joyce , Zachariah Cherian , De Freitas Duarte Mota , Stubbs Evan B. , Ramasamy Ravichandran , Castro M. Margarida C. A. , Geraldes Carlos F. G. C.
Published: July 2010

Abstract: Lithium has been used clinically in the treatment of manic depression. However, its pharmacologic mode of action remains unclear. Characteristics of Li+ interactions in red blood cells (RBCs) have been identified. We investigated Li+ interactions on human neuroblastoma SH‐SY5Y cells by developing a novel 7Li NMR method that provided a clear estimation of the intra‐ and extracellular amounts of Li+ in the presence of the shift reagent thulium‐1,4,7,10‐tetrazacyclododecane‐N,N′,N″,N‴‐tetramethylene phosphonate (HTmDOTP4−). The first‐order rate constants of Li+ influx and efflux for perfused, agarose‐embedded SH‐SY5Y cells in the presence of 3 mM HTmDOTP4− were 0.055 ± 0.006 (n = 4) and −0.025 ± 0.006 min−1 (n = 3), respectively. Significant increases in the rate constants of Li+ influx and efflux in the presence of 0.05 mM veratridine indicated the presence of Na+ channel‐mediated Li+ transport in SH‐SY5Y cells. 7Li NMR relaxation measurements showed that Li+ is immobilized more in human neuroblastoma SH‐SY5Y cells than in human RBCs.
Characterization of Granular Particles Isolated from Postsynaptic Densities
Authors: Lai Sun‐Ling , Ling Shuo‐Chien , Kuo Ling‐Ho , Shu Yu‐Chung , Chow Wei‐Yuan , Chang Yen‐Chung
Published: July 2010

Abstract: We describe here the isolation and biochemical characterization of a population of protein aggregates from the postsynaptic density (PSD) prepared from pig cerebral cortex. The protein constituents of these aggregates are linked together primarily by disulfide bonds. Negative staining electron microscopy revealed that the isolated protein aggregates were granular objects with an average outside diameter of ∼21 nm and with small protrusions on their surface. The major constituents of the isolated granular aggregates consist of tubulin and an unidentified protein of 70 kDa in size. Small amounts of the α subunit of calcium/calmodulin‐dependent protein kinase II and subunits of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid and NMDA subtypes of glutamate receptors were also detected by immunoblotting. Actin, however, was not found in these granular aggregates. We propose that these granular protein aggregates correspond to the ∼20‐nm‐diameter granular particles of the PSD on the basis of their biochemical and morphological characteristics. The spatial arrangement of these granular aggregates relative to other components of the postsynaptic terminal is also postulated here.
Alternative Splicing at the 3′‐cDNA of Human Tryptophan Hydroxylase
Authors: Wang Guo‐An , Coon Steven L. , Kaufman Seymour
Published: July 2010

Abstract: Two alternatively spliced transcripts of human tryptophan hydroxylase (TPH) were identified that differed at the 3′ end of the open reading frame. Comparison of the human TPH cDNA and genomic sequences revealed that an intron containing an in‐frame stop codon could be alternatively spliced out of intron 11. This splicing would give rise to two human TPH isoforms with different C termini; the one that derives from the nonspliced intron contains a putative cyclic AMP‐dependent protein kinase site, whereas the other one, which is 22 amino acids longer, does not. Analysis of various human tissues by RT‐PCR revealed that the spliced TPH mRNA species was detected in all the postmortem tissues we tested, but the nonspliced species was expressed in only some tissues.
Persistent Expression of Fas/FasL mRNA in the Mouse Hippocampus After a Single NMDA Injection
Authors: Shin Song‐Woo , Park Jong‐Wook , Suh Min‐Ho , Suh Sung‐Il , Choe Byung‐Kil
Published: July 2010

Abstract: Synaptic reorganization plays a very important role in brain adaptations to environmental stimuli, diseases, and aging processes. The NMDA model of excitotoxic injury was used to investigate the long‐term molecular changes in the surviving neural cells in the mouse hippocampus. We demonstrated that a single intraperitoneal injection of NMDA produces persistent expression of c‐fos, c‐jun, Fas, and Fas ligand (FasL) mRNA in the hippocampus for 5 months. To determine the cellular origin of those gene transcripts in our in vivo model, a glial cell line and primary fetal neuronal culture were used to investigate the inducibility of the c‐fos, c‐jun, Fas, and FasL mRNA by NMDA. Both c‐fos and Fas mRNA expression was observed in the NMDA‐treated glial or neuronal cultures; however, c‐jun and FasL mRNA was undetectable in this study. In our in vivo model, mossy fiber sprouting and apoptosis were also observed up to 40 days after the NMDA injection. Therefore, we hypothesize that the observed long‐term expression of c‐fos, c‐jun, Fas, and FasL mRNAs may reflect the ongoing synaptic reorganization.
Hormonal Regulation of the mRNA Encoding the Ketogenic Enzyme Mitochondrial 3‐Hydroxy‐3‐Methylglutaryl‐CoA Synthase in Neonatal Primary Cultures of Cortical Astrocytes and Meningeal Fibroblasts
Authors: Cullingford Tim E. , Bhakoo Kishore K. , Clark John B.
Published: July 2010

Abstract: We have previously identified cerebellum to contain significantly higher levels, compared with other brain regions, of the mRNA encoding the key ketogenic enzyme mitochondrial 3‐hydroxy‐3‐methylglutaryl‐CoA synthase (mHS). In this report, we extend these observations, using primary cultures of cerebellar astrocytes and cerebellar granule neurons, and show that mHS mRNA was not readily detected in these cell types, suggesting that other cerebellar cell types account for mHS mRNA abundances observed in cerebellum. In contrast, we report, for the first time, the ready detection of mHS mRNA together with the mRNAs encoding the remaining enzymes of the 3‐hydroxy‐3‐methylglutaryl‐CoA cycle, namely, mitochondrial acetoacetyl‐CoA thiolase and 3‐hydroxy‐3‐methylglutaryl‐CoA lyase, in primary cultures of neonatal meningeal fibroblasts. Based on observations of the effects of fetal calf serum in the culture medium and the documented effects of various hormones on mHS mRNA levels in liver, we show that the glucocorticoid hydrocortisone effects a selective fourfold increase in mHS mRNA abundances in both neonatal meningeal fibroblasts and neonatal cortical astrocytes cultured in a serum‐free/hormone‐free medium.
Pituitary Adenylyl Cyclase‐Activating Polypeptide and Nerve Growth Factor Use the Proteasome to Rescue Nerve Growth Factor‐Deprived Sympathetic Neurons Cultured From Chick Embryos
Authors: Przywara Dennis A. , Kulkarni Jayant S. , Wakade Taruna D. , Leontiev Dmitry V. , Wakade Arun R.
Published: July 2010

Abstract: Removal of nerve growth factor (NGF) from sympathetic neurons initiates a neuronal death program and apoptosis. We show that pituitary adenylyl cyclase‐activating polypeptide (PACAP) prevents apoptosis in NGF‐deprived sympathetic neurons. PACAP (100 nM) added to culture medium at the time of plating failed to support neuronal survival. However, in neurons grown for 2 days with NGF and then deprived of NGF, PACAP prevented cell death for the next 24–48 h. Uptake of [3H]norepinephrine ([3H]NE) was used as an index of survival and decreased >50% in NGF‐deprived cultures within 24 h. PACAP (1–100 nM) restored [3H]NE uptake to 92 ± 8% of that of NGF‐supported controls. Depolarization‐induced [3H]NE release in neurons rescued by PACAP was the same as that in NGF‐supported neurons. PACAP rescue was not mimicked by forskolin or 8‐bromo‐cyclic AMP and was not blocked by the protein kinase A inhibitor Rp‐adenosine 3′,5′‐cyclic monophosphothioate. Mobilization of phosphatidylinositol by muscarine failed to support NGF‐deprived neurons. Thus, PACAP may use novel signaling to promote survival of sympathetic neurons. The apoptosis‐associated caspase CPP32 activity increased approximately fourfold during 6 h of NGF withdrawal (145 ± 40 versus 38 ± 17 nmol of substrate cleaved/min/mg of protein) and returned to even below the control level in NGF‐deprived, PACAP‐rescued cultures (14 ± 7 nmol/min/mg of protein). Readdition of NGF or PACAP to NGF‐deprived cultures reversed CPP32 activation, and this was blocked by lactacystin, a potent and specific inhibitor of the 20S proteasome, suggesting that NGF and PACAP target CPP32 for destruction by the proteasome. As PACAP is a preganglionic neurotransmitter in autonomic ganglia, we propose a novel function for this transmitter as an apoptotic rescuer of sympathetic neurons when the supply of NGF is compromised.
Inhibition of Phosphatidylinositol 3‐Kinase Activity Blocks Cellular Differentiation Mediated by Glial Cell Line‐Derived Neurotrophic Factor in Dopaminergic Neurons
Authors: Pong Kevin , Xu Ren Y. , Baron Will F. , Louis Jean‐Claude , Beck Klaus D.
Published: July 2010

Abstract: Glial cell line‐derived neurotrophic factor (GDNF) is a potent survival factor for midbrain dopaminergic neurons. To begin to understand the intracellular signaling pathways used by GDNF, we investigated the role of phosphatidylinositol 3‐kinase activity in GDNF‐stimulated cellular function and differentiation of dopaminergic neurons. We found that treatment of dopaminergic neuron cultures with 10 ng/ml GDNF induced maximal levels of Ret phosphorylation and produced a profound increase in phosphatidylinositol 3‐kinase activity, as measured by western blot analysis and lipid kinase assays. Treatment with 1 µM 2‐(4‐morpholinyl)‐8‐phenylchromone (LY294002) or 100 nM wortmannin, two distinct and potent inhibitors of phosphatidylinositol 3‐kinase activity, completely inhibited GDNF‐induced phosphatidylinositol 3‐kinase activation, but did not affect Ret phosphorylation. Furthermore, we examined specific biological functions of dopaminergic neurons: dopamine uptake activity and morphological differentiation of tyrosine hydroxylase‐immunoreactive neurons. GDNF significantly increased dopamine uptake activity and promoted robust morphological differentiation. Treatment with LY294002 completely abolished the GDNF‐induced increases of dopamine uptake and morphological differentiation of tyrosine hydroxylase‐immunoreactive neurons. Our findings show that GDNF‐induced differentiation of dopaminergic neurons requires phosphatidylinositol 3‐kinase activation.
Regulation of Plasminogen Activator Inhibitor‐1 mRNA Accumulation by Basic Fibroblast Growth Factor and Transforming Growth Factor‐β1 in Cultured Rat Astrocytes
Authors: Treichel Julia A. , Reddington Martin , Kreutzberg Georg W.
Published: July 2010

Abstract: The effects of transforming growth factor‐β1 (TGF‐β1) and basic fibroblast growth factor (bFGF) were examined on the accumulation of plasminogen activator inhibitor‐1 (PAI‐1) mRNA in astrocytes in vitro. Both cytokines stimulated PAI‐1 mRNA expression transiently with a maximal fivefold (bFGF) and 30‐fold (TGF‐β1) at 4 h, decreasing to basal levels within 32 h. EC50 values were 1.4 nM for bFGF and 6.7 pM for TGF‐β1 on PAI‐1 mRNA accumulation. A twofold increase in content of tPA mRNA was observed with bFGF but not with TGF‐β1. The action of TGF‐β1 on PAI‐1 mRNA was inhibited by cycloheximide, indicating a requirement for de novo protein synthesis. In contrast, cycloheximide potentiated the action of bFGF. Nuclear run‐on assays showed that bFGF, but not TGF‐β1, stimulated astrocytic PAI‐1 gene transcription. Thus, TGF‐β1 predominantly uses posttranscriptional mechanisms to raise the level of PAI‐1 mRNA in astrocytes, whereas bFGF acts at both the transcriptional and posttranscriptional levels. The data reveal differences in the mechanisms underlying the regulation of PAI‐1 mRNA levels by TGF‐β1 in astrocytes compared with other cells. The action of TGF‐β1 and bFGF on the plasminogen activator system in astrocytes might be involved in the cellular events accompanying glial activation following injury of the CNS.
The Human N‐Methyl‐d‐Aspartate Receptor 2C Subunit: Genomic Analysis, Distribution in Human Brain, and Functional Expression
Authors: Daggett L. P. , Johnson E. C. , Varney M. A. , Lin F.‐F. , Hess S. D. , Deal C. R. , Jachec C. , Lu C.‐C. , Kerner J. A. , Landwehrmeyer G. B. , Standaert D. G. , Young A. B. , Harpold M. M. , Veliçelebi G.
Published: July 2010

Abstract: cDNAs encoding four isoforms of the human NMDA receptor (NMDAR) NMDAR2C (hNR2C‐1, ‐2, ‐3, and ‐4) have been isolated and characterized. The overall identity of the deduced amino acid sequences of human and rat NR2C‐1 is 89.0%. The sequences of the rat and human carboxyl termini (Gly925‐Val1,236) are encoded by different exons and are only 71.5% homologous. In situ hybridization in human brain revealed the expression of the NR2C mRNA in the pontine reticular formation and lack of expression in substantia nigra pars compacta in contrast to the distribution pattern observed previously in rodent brain. The pharmacological properties of hNR1A/2C were determined by measuring agonist‐induced inward currents in Xenopus oocytes and compared with those of other human NMDAR subtypes. Glycine, glutamate, and NMDA each discriminated between hNR1A/2C‐1 and at least one of hNR1A/2A, hNR1A/2B, or hNR1A/2D subtypes. Among the antagonists tested, CGS 19755 did not significantly discriminate between any of the four subtypes, whereas 5,7‐dichlorokynurenic acid distinguished between hNR1A/2C and hNR1A/2D. Immunoblot analysis of membranes isolated from HEK293 cells transiently transfected with cDNAs encoding hNR1A and each of the four NR2C isoforms indicated the formation of heteromeric complexes between hNR1A and all four hNR2C isoforms. HEK293 cells expressing hNR1A/2C‐3 or hNR1A/2C‐4 did not display agonist responses. In contrast, we observed an agonist‐induced elevation of intracellular free calcium and whole‐cell currents in cells expressing hNR1A/2C‐1 or hNR1A/2C‐2. There were no detectable differences in the macroscopic biophysical properties of hNR1A/2C‐1 or hNR1A/2C‐2.
The Neurosteroid Pregnenolone Sulfate Increases Cortical Acetylcholine Release: A Microdialysis Study in Freely Moving Rats
Authors: Darnaudéry Muriel , Koehl Muriel , Pallarés Marc , Le Moal Michel , Mayo Willy
Published: July 2010

Abstract: The effects of pregnenolone sulfate (Preg‐S) administrations (0, 12, 48, 96, and 192 nmol intracerebroventricularly) on acetylcholine (ACh) release in the frontal cortex and dorsal striatum were investigated by on‐line microdialysis in freely moving rats. Following Preg‐S administration, extracellular ACh levels in the frontal cortex increased in a dose‐dependent manner, whereas no change was observed in the striatum. The highest doses (96 and 192 nmol) induced a threefold increase above control values of ACh release, the intermediate dose of 48 nmol led to a twofold increase, whereas after the dose of 12 nmol, the levels of ACh were not different from those observed after vehicle injection. The increase in cortical ACh reached a maximum 30 min after administration for all the active doses. Taken together, these results suggest that Preg‐S interacts with the cortical cholinergic system, which may account, at least in part, for the promnesic and/or antiamnesic properties of this neurosteroid.
Increased Nuclear DNA Oxidation in the Brain in Alzheimer's Disease
Authors: Gabbita S. Prasad , Lovell Mark A. , Markesbery William R.
Published: July 2010

Abstract: Multiple lines of evidence indicate that oxidative stress is a contributor to neuronal death in Alzheimer's disease (AD). The oxidative damage that occurs to DNA may play a role in both normal aging and neurodegenerative diseases, including AD. This is a study of the oxidative damage that occurs in nuclear DNA in the brains of AD patients and cognitively intact, prospectively evaluated, age‐matched control subjects. Nuclear DNA from frontal, temporal, and parietal lobes and cerebellum was isolated from 11 control subjects and 9 AD subjects, and oxidized purine and pyrimidine bases were quantitated using gas chromatography/mass spectrometry. Stable isotope‐labeled oxidized base analogues were used as internal standards to measure 5‐hydroxyuracil, 5‐hydroxycytosine, 8‐hydroxyadenine, 4,6‐diamino‐5‐formamidopyrimidine (Fapy‐adenine), 8‐hydroxyguanine, and 2,6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine (Fapy‐guanine). Statistically significant elevations of 5‐hydroxycytosine, 5‐hydroxyuracil, 8‐hydroxyadenine, and 8‐hydroxyguanine were found in AD brain compared with control subjects (p < 0.05). There was an increased trend in the levels of Fapy‐adenine in the AD brain, and Fapy‐guanine showed a trend toward higher levels in control brains compared with AD. A generally higher level of oxidative DNA damage was present in neocortical regions than cerebellum. No significant correlation was observed between the oxidized bases and neurofibrillary tangle and senile plaque counts. Our results demonstrate that nuclear DNA damage by oxygen‐derived radicals is increased in AD and support the concept that the brain is under increased oxidative stress in AD.
Brain Mitochondria Catalyze the Oxidation of 7‐(2‐Aminoethyl)‐3,4‐Dihydro‐5‐Hydroxy‐2H‐1,4‐Benzothiazine‐3‐Carboxylic Acid (DHBT‐1) to Intermediates that Irreversibly Inhibit Complex I and Scavenge Glutathione: Potential Relevance to the Pathogenesis of Parkinson's Disease
Authors: Li Hong , Shen Xue‐Ming , Dryhurst Glenn
Published: July 2010

Abstract: We have proposed that a very early step in the pathogenesis of idiopathic Parkinson's disease is the elevated translocation of l‐cysteine into neuromelanin‐pigmented dopaminergic neurons in the substantia nigra. This influx of l‐cysteine was proposed to divert the normal neuromelanin pathway by scavenging dopamine‐o‐quinone, formed by autoxidation of cytoplasmic dopamine, to give initially 5‐S‐cysteinyldopamine, which is further oxidized to 7 ‐ (2 ‐ aminoethyl) ‐ 3,4 ‐ dihydro ‐ 5 ‐ hydroxy ‐ 2H ‐ 1,4 ‐ benzothiazine‐3‐carboxylic acid (DHBT‐1). In a recent report, it was demonstrated that DHBT‐1 evokes inhibition of complex I respiration when incubated with intact rat brain mitochondria and a time‐dependent irreversible inhibition of NADH‐coenzyme Q1 (CoQ1) reductase when incubated with mitochondrial membranes. In this study, it is established that the time dependence of NADH‐CoQ1 reductase inhibition reflects the oxidation of DHBT‐1, catalyzed by an unknown constituent of the inner mitochondrial membrane, to an o‐quinone imine intermediate that rearranges to 7‐(2‐aminoethyl) ‐ 5 ‐ hydroxy ‐ 1,4 ‐ benzothiazine ‐ 3 ‐ carboxylic acid (BT‐1) and decarboxylates to 7‐(2‐aminoethyl)‐5‐hydroxy‐1,4‐benzothiazine (BT‐2), which are further catalytically oxidized to o‐quinone imine intermediates. The electrophilic o‐quinone imine intermediates formed in these mitochondria‐catalyzed oxidations of DHBT‐1, BT‐1, and BT‐2 are proposed to bind covalently to key sulfhydryl residues at the complex I site, thus evoking irreversible inhibition of NADH‐CoQ1 reductase. Evidence for this mechanism derives from the fact that greater than equimolar concentrations of glutathione completely block inhibition of NADH‐CoQ1 reductase by DHBT‐1, BT‐1, and BT‐2 by scavenging their electrophilic o‐quinone imine metabolites to form glutathionyl conjugates. The results of this investigation may provide insights into the irreversible loss of glutathione and decreased mitochondrial complex I activity, which are both anatomically specific to the substantia nigra and exclusive to Parkinson's disease.
Mitogen‐Activated Protein Kinase‐Dependent and Protein Kinase C‐Dependent Pathways Link the m1 Muscarinic Receptor to β‐Amyloid Precursor Protein Secretion
Authors: Haring Rachel , Fisher Abraham , Marciano Daniele , Pittel Zipora , Kloog Yoel , Zuckerman Avi , Eshhar Nomi , Heldman Eliahu
Published: July 2010

Abstract: Full and functionally selective M1 muscarinic agonists (carbachol and AF102B, respectively) activate secretion of the soluble form of amyloid precursor protein (APPs) in PC12 cells expressing the m1 muscarinic receptor (PC12M1 cells). This activation is further augmented by neurotrophins such as nerve growth factor and basic fibroblast growth factor. Muscarinic stimulation activates two transduction pathways that lead to APPs secretion: protein kinase C (PKC)‐dependent and mitogen‐activated protein kinase (MAPK)‐dependent pathways. These pathways operate in parallel and converge with transduction pathways of neurotrophins, resulting in enhancement of APPs secretion when both muscarinic agonist and neurotrophins stimulate PC12M1 cells. These conclusions are supported by the following findings: (a) Only partial blockade of APPs secretion is observed when PKC, p21ras, or MAPK is fully inhibited by their respective specific inhibitors, GF109203X, S‐trans,trans‐farnesylthiosalicylic acid, and PD98059. (b) K252a, which blocks PKC and phorbol 12‐myristate 13‐acetate‐induced APPs secretion, enhances both muscarinic‐stimulated MAPK activation and APPs secretion. (c) Activation of MAPK in PC12M1 cells by muscarinic agonists is Ras‐dependent but PKC‐independent and is enhanced synergistically by neurotrophins. These results suggest that muscarinic stimulation of APPs secretion is mediated by at least two independent pathways that converge and enhance the signal for APPs secretion at the convergence point.
Recycling of Carbon into Lipids Synthesized De Novo Is a Quantitatively Important Pathway of α‐[U‐13C]Linolenate Utilization in the Developing Rat Brain
Authors: Menard Chantale R. , Goodman Keith J. , Corso Thomas N. , Brenna J. Thomas , Cunnane Stephen C.
Published: July 2010

Abstract: Docosahexaenoate is important for normal neural development. It can be derived from α‐linolenate, but carbon from α‐linolenate is also recycled into de novo lipid synthesis. The objective of this study was to quantify the amount of α‐linolenate used to produce docosahexaenoate versus lipids synthesized de novo that accumulate in the brain of the developing rat. A physiological dose of carbon‐13‐labeled α‐linolenate was injected into the stomachs of mother‐reared 6‐day‐old rat pups. Total lipids of brain, liver, and gut were extracted from rats killed 3 h to 30 days after dosing. Carbon‐13 enrichment was determined by isotope ratio mass spectrometry. Carbon‐13‐enriched α‐linolenate was not detected in the brain at any time point, and its levels in liver and gut exceeded detection limits at most time points, so tracer mass was quantified mainly for three end products—docosahexaenoate, palmitate, and cholesterol. Carbon‐13‐enriched cholesterol, palmitate, docosahexaenoate, and water‐soluble metabolites were detected in brain, liver, and gut. Enrichment (in micrograms of carbon‐13 per organ) in brain cholesterol exceeded that in brain docosahexaenoate by four‐ to 16‐fold over the duration of the study. Enrichment in brain palmitate exceeded that in brain docosahexaenoate by three‐ to 30‐fold over the first 8 days of the study. These results indicate that carbon from α‐linolenate is not exclusively conserved for synthesis of longer n‐3 polyunsaturates but is a readily accessible carbon source for de novo lipogenesis during early brain development in the suckling rat. Owing to a high rate of β‐oxidation and carbon recycling, dependence on α‐linolenate as the sole source of docosahexaenoate may incur a potential risk of providing insufficient docosahexaenoate for the developing brain.
Association of HAP1 Isoforms with a Unique Cytoplasmic Structure
Authors: Li Shi‐Hua , Gutekunst Claire‐Anne , Hersch Steven M. , Li Xiao‐Jiang
Published: July 2010

Abstract: HAP1 is a neural protein and interacts with the Huntington's disease protein huntingtin. There are at least two HAP1 isoforms, HAP1‐A and HAP1‐B, which have different C‐terminal amino acid sequences. Here we report that both HAP1 isoforms associate with a unique cytoplasmic structure in neurons of rat brain. The HAP1‐immunoreactive structure appears as an inclusion that is an oval mass of electron‐dense material, 0.5–3 µm in diameter, containing many curvilinear or ring‐shaped segments, and often containing electron‐lucent cores. This structure is very similar to those previously termed the stigmoid body, nematosome, or botrysome. Transfection of cell lines with cDNA encoding HAP1‐A, but not HAP1‐B, resulted in similar HAP1‐immunoreactive inclusions in the cytoplasm, suggesting that HAP1‐A is essential to the formation of this structure. Yeast two‐hybrid and transfection studies show that both HAP1‐A and HAP1‐B can self‐associate, implying that native HAP1 in the cytoplasmic inclusion may be a heteromultimer of HAP1‐A and HAP1‐B. Coexpression of HAP1‐A and HAP1‐B in human embryonic kidney 293 cells demonstrates that the ratio of the expressed HAP1‐A to HAP1‐B regulates the formation of HAP1‐associated inclusions. We propose that HAP1 isoforms are involved in the formation of HAP1‐immunoreactive inclusions in the neuronal cytoplasm.
Cyclic AMP Regulation of Giα2 and Giα3 mRNAs and Proteins in Astroglial Cells
Authors: El Jamali Amina , Rachdaoui Nadia , Dib Karim , Corrèze Claude
Published: July 2010

Abstract: Culture of rat astrocytes in the presence of 10 µM forskolin, isoproterenol, a nonselective β‐adrenergic agonist, or 8‐bromo‐cyclic AMP increased transiently in a time‐dependent manner the levels of Giα2 and Giα3 mRNAs as measured by northern blot analysis. Giα1 mRNA was not expressed in these cells. After 6–9 h of culture with forskolin or isoproterenol the amounts of Giα2 and Giα3 mRNAs were maximal (150–300% over control). In cells challenged with 8‐bromo‐cyclic AMP, the level of Giα2 mRNA level was maximal after 1 h of culture, while the maximal for Giα3 mRNA was reached after 6 h of culture. For prolonged exposure times (24 h) to these agents the levels of Giα2 and Giα3 mRNAs decreased to the values observed in control cells. The forskolin‐induced increase in Giα2 protein expression measured by western blot analysis was similar to the increase in Giα2 mRNA amount. In contrast, forskolin induced only a modest increase in the quantity of Giα3 protein (150% over control) despite a large increase of Giα3 mRNA content. Transcription rates and RNA stability were measured simultaneously after isolation of newly synthesized mRNA was performed. The half‐lives of Giα mRNAs were ∼0.7 and 3 h for Giα2 and Giα3, respectively. Culturing astrocytes in the presence of forskolin resulted in an increase in the half‐lives of Giα2 mRNA and Giα3 mRNA by five‐ and twofold, respectively. The relative transcription rate of the Giα3 gene was slightly increased by ∼1.3–1.5‐fold in forskolin‐treated cells but not that of Giα2 gene. These results suggest that cyclic AMP enhanced Giα2 and Giα3 mRNA expression by both transcriptional and posttranscriptional mechanisms, with an increase of Giα mRNA stability as one of the major checkpoints.
Possible Involvement of Mitogen‐Activated Protein Kinase in Phospholipase D Activation Induced by H2O2, but Not by Carbachol, in Rat Pheochromocytoma PC12 Cells
Authors: Ito Yuzuru , Nakashima Shigeru , Nozawa Yoshinori
Published: July 2010

Abstract: We have previously reported that hydrogen peroxide (H2O2) induced a considerable increase of phospholipase D (PLD) activity and phosphorylation of mitogen‐activated protein (MAP) kinase in PC12 cells. H2O2‐induced PLD activation and MAP kinase phosphorylation were dose‐dependently inhibited by a specific MAP kinase kinase inhibitor, PD 098059. In contrast, carbachol‐mediated PLD activation was not inhibited by the PD 098059 pretreatment whereas MAP kinase phosphorylation was prevented. These findings indicated that MAP kinase is implicated in the PLD activation induced by H2O2, but not by carbachol. In the present study, H2O2 also caused a marked release of oleic acid (OA) from membrane phospholipids in PC12 cells. As we have previously shown that OA stimulates PLD activity in PC12 cells, the mechanism of H2O2‐induced fatty acid liberation and its relation to PLD activation were investigated. Pretreatment of the cells with methylarachidonyl fluorophosphonate (MAFP), a phospholipase A2 (PLA2) inhibitor, almost completely prevented the release of [3H]OA by H2O2 treatment. From the preferential release of OA and sensitivity to other PLA2 inhibitors, the involvement of a Ca2+‐independent cytosolic PLA2‐type enzyme was suggested. In contrast, to OA release, MAFP did not inhibit PLD activation by H2O2. The inhibitory profile of the OA release by PD 098059 did not show any correlation with that of MAP kinase. These results lead us to suggest that H2O2‐induced PLD activation may be mediated by MAP kinase and also that H2O2‐mediated OA release, which would be catalyzed by a Ca2+‐independent cytosolic PLA2‐like enzyme, is not linked to the PLD activation in PC12 cells.