bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphatase Resistance of ERK2 Brain Kinase PK40erk2
Authors: Roder Hanno M. , Hoffman Frederick J. , Schröder Werner
Published: July 2010

Abstract: We have previously shown that a brain protein kinase, termed PK40, catalyzes the multiple phosphorylation of the KSP‐repeat site of neurofilaments (NFs) and also can transform τ proteins into the paired helical filament‐like state as found in Alzheimer's disease (AD) brains. Protein sequence analysis suggests that PK40 is a form of the extracellular signal‐regulated kinase ERK2. A subpopulation of ERK2 species in soluble brain fractions can be efficiently phosphorylated and activated in cell‐free systems, simply by adding Mg2+‐ATP. Two phosphoisoforms of PK40erk2 are formed in this process, which have a reduced gel mobility, very much like the ERK2 form obtained in cell culture by stimulation with growth factors. One of these low‐mobility forms cannot be inactivated with protein phosphatase 2A (PP2A) or with tyrosine phosphatases. The second form can be slowly inactivated by PP2A. In this case two Ser/Thr phosphates are removed at different rates during inactivation: One phosphate is very quickly removed to result in the formation of a high‐mobility 39‐kDa ERK2 species without consequence for activity; the other, slowly removed Ser/Thr phosphate controls the activity but has no effect on the gel mobility of ERK2. These results show that forms of ERK2 exist with properties different from the previously characterized ERK2 (p42mapk) from stimulated cell cultures. The active ERK2 forms produced in the presence of Mg2+‐ATP alone could provide an explanation for the existence of constitutive ERK2‐like NF phosphorylation in vivo. Excessive formation of an ERK2 species resistant to inactivation by PP2A might be relevant to the persistent pathological τ hyperphosphorylation in AD.
Characterization of a Novel Phospholipase A2 Activity in Human Brain
Authors: Ross Brian M. , Kim Dae K. , Bonventre Joseph V. , Kish Stephen J.
Published: July 2010

Abstract: Phospholipases A2 (PLA2) are a family of enzymes that catalyze the removal of fatty acid residues from phosphoglycerides. The enzyme is postulated to be involved in several human brain disorders, although little is known regarding the status of PLA2 activity in human CNS. We therefore have characterized some aspects of the PLA2 activity present in the temporal cortex of human brain. More PLA2 activity was found in the membrane (particulate) fraction than in the cytosolic fraction. The enzyme could be solubilized from particulate material using 1 M potassium chloride, and was capable of hydrolyzing choline phosphoglyceride (CPG) and ethanolamine phosphoglyceride (EPG), with a preference (approximately eightfold) for EPG over CPG. When the solubilized particulate enzyme was subjected to gel filtration chromatography, PLA2 activity eluted in a high molecular mass fraction (∼180 kDa). PLA2 activity was weakly stimulated by dithiothreitol, strongly stimulated by millimolar concentrations of calcium ions, and inhibited by brief heat treatment at 57°C, bromophenacyl bromide, the arachidonic acid derivative AACOCF3, γ‐linolenoyl amide, and N‐methyl γ‐linolenoyl amide. Thus, whereas the human brain enzyme(s) characterized in our study displays some of the characteristics of previously characterized PLA2s, it differs in several key features.
A Novel cDNA Sequence Encoding the Precursor of the d‐Amino Acid‐Containing Neuropeptide Fulicin and Multiple α‐Amidated Neuropeptides from Achatina fulica
Authors: Yasuda‐Kamatani Yoshimi , Nakamura Masahiko , Minakata Hiroyuki , Nomoto Kyosuke , Sakiyama Fumio
Published: July 2010

Abstract: Fulicin (Phe‐d‐Asn‐Glu‐Phe‐Val‐NH2) is an endogenous neuropeptide containing a d‐amino acid from ganglia of the African giant snail Achatina fulica. We have cloned a novel cDNA (1,995 nucleotides) encoding a fulicin precursor from the snail cerebral and subesophageal ganglia. The fulicin precursor protein (357 amino acids) contains one copy of fulicin and at least nine other putative α‐amidated neuropeptides composed of four to six amino acid residues. Seven of the nine neuropeptides were novel, and the other two had the same structures as Mytilus inhibitory peptide‐related peptides previously isolated from the ganglia of Helix pomatia. All sequences of 10 peptides are flanked by Lys‐Arg(Lys) at the N‐terminus and by Gly‐Lys‐Arg(Lys) at the C‐terminus. Nucleotide sequence analysis revealed that d‐Asn present in fulicin is encoded by the usual l‐Asn codon (AAT). Although fulicin has as yet only been isolated from the central ganglia, RNA blot analysis revealed that single transcripts of ∼2.0 kb in size also exist in the ventricles and atria. These results suggest that fulicin and related peptides are produced in neurons and the heart by the processing of a ribosomally made precursor, although the mechanism of in‐chain epimerization remains unclear.
7B2 Is a Specific Intracellular Binding Protein of the Prohormone Convertase PC2
Authors: Benjannet Suzanne , Savaria Diane , Chrétien Michel , Seidah Nabil G.
Published: July 2010

Abstract: Biosynthetic pulse‐chase analyses have previously demonstrated that the prohormone convertase PC2 is first synthesized as a precursor pro‐PC2 and that zymogen activation to PC2 occurs following the slow exit of pro‐PC2 from the endoplasmic reticulum (ER) and its concentration within the trans‐Golgi network (TGN). The endocrine and neural protein 7B2 is first synthesized as a nonglycosylated precursor (pro‐7B2), which is cleaved within the TGN by a furin‐like ubiquitous convertase at the RRKRR155S site to generate 7B2. In this report, we demonstrate that within the ER, pro‐7B2 binds pro‐PC2 but not any of the other convertases furin, PC1, PACE4, or PC5. This specific binding is Ca2+ dependent and does not require an N‐glycosylated pro‐PC2. Mutagenesis of the RRKRRS sequence demonstrated that the intact hexapeptide is critical for this binding, because the latter was abolished by mutations of the RR152 and greatly diminished by mutations of either the R151 or S156 residues of pro‐7B2. Once the complex is formed in the ER, it is then transported to the TGN where furin or a furin‐like convertase cleaves both precursors, even when present as a complex. We also provide evidence that following zymogen cleavage, 7B2 remains bound to PC2, suggesting the presence of at least one other Ca2+‐dependent binding site within the 7B2 sequence. Coexpression of 7B2 and PC2, although resulting in an elevation of the level of pro‐PC2, did not eliminate the processing of pro‐PC2 to PC2. Accordingly, cellular coexpression of 7B2 together with PC2 and proopiomelanocortin only marginally diminished the ability of PC2 to cleave proopiomelanocortin into β‐endorphin in constitutive cells and had no effect in regulated cells. These results suggest that in vivo pro‐7B2 is a specific PC2‐binding protein that only transiently inhibits the processing of pro‐PC2 until it reaches the TGN.
Exacerbation of NMDA, AMPA, and l‐Glutamate Excitotoxicity by the Succinate Dehydrogenase Inhibitor Malonate
Authors: Greene James G. , Greenamyre J. Timothy
Published: July 2010

Abstract: We report that a subtoxic dose of the succinate dehydrogenase (SDH) inhibitor malonate greatly enhances the neurotoxicity of three different excitatory amino acid agonists: N‐methyl‐d‐aspartate (NMDA), S‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (S‐AMPA), and l‐glutamate. In male Sprague‐Dawley rats, intrastriatal stereotaxic injection of malonate alone (0.6 µmol), NMDA alone (15 nmol), S‐AMPA alone (1 nmol), or glutamate alone (0.6 µmol) produced negligible toxicity as assessed by measurement of lesion volume. Coinjection of subtoxic malonate with NMDA produced a large lesion (15.2 ± 1.4 mm3), as did coinjection of malonate with S‐AMPA (11.0 ± 1.0 mm3) or glutamate (12.8 ± 0.7 mm3). Administration of the noncompetitive NMDA antagonist MK‐801 (5 mg/kg i.p.) completely blocked the toxicity of malonate plus NMDA (0.5 ± 0.3 mm3). This dose of MK‐801 had little effect on the lesion produced by malonate plus S‐AMPA (9.0 ± 0.7 mm3), but it attenuated the toxicity of malonate plus glutamate by ∼40% (7.5 ± 0.9 mm3). Coinjection of the AMPA antagonist 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoylbenzo(f)‐quinoxaline (NBQX; 2 nmol) had no effect on malonate plus NMDA or malonate plus glutamate toxicity (12.3 ± 1.8 and 14.0 ± 0.9 mm3, respectively) but greatly attenuated malonate plus S‐AMPA toxicity (1.5 ± 0.9 mm3). Combination of the two antagonists conferred no additional neuroprotection in any paradigm. These results indicate that metabolic inhibition exacerbates both NMDA receptor‐ and non‐NMDA receptor‐mediated excitotoxicity. They also suggest that the NMDA receptor may play a major role in situations of metabolic compromise in vivo, where glutamate is the endogenous agonist. Furthermore, glutamate toxicity under conditions of metabolic compromise may not be mediated entirely by ionotropic glutamate receptors.
Inhibition of Nitric Oxide Synthase Activity Attenuates Striatal Malonate Lesions in Rats
Authors: Maragos William F. , Silverstein Faye S.
Published: July 2010

Abstract: Mitochondrial inhibitors such as malonate are potent neurotoxins in vivo. Intrastriatal injections of malonate result in neuronal damage reminiscent of “excitotoxic” lesions produced by compounds that activate NMDA receptors. Although the mechanism of cell death produced by malonate is uncertain, overactivation of NMDA receptors may be involved; pretreatment of animals with NMDA antagonists provides neuroprotection against malonate lesions. NMDA receptor activation stimulates the enzyme nitric oxide (NO) synthase (NOS). Elevated tissue levels of NO may generate highly reactive intermediates that impair mitochondrial function. We hypothesized that NO may be a mediator of malonate toxicity. We investigated whether in vivo inhibition of NO production by the NOS inhibitor Nω‐nitro‐l‐arginine (NLA) would attenuate lesions produced by intrastriatal injections of malonate. We found that systemic injections of 3 mg/kg of NLA significantly reduced the extent of histologic damage elicited by intrastriatal injections of 1.5 µmol of malonate in adult rats.
Molecular Cloning and Tissue Expression of a cDNA Encoding IP1—A P0‐Like Glycoprotein of Trout CNS Myelin
Authors: Stratmann A. , Jeserich G.
Published: July 2010

Abstract: A full‐length cDNA encoding a major structural glycoprotein of trout CNS myelin (IP1) was cloned and sequenced. The deduced amino acid sequence exhibited a significant structural homology with the P0 proteins of rat PNS and shark CNS. Sequence conservation was strongest in the extracellular domain, and it included the position of the two cysteine residues required for stabilization of an immunoglobulin‐like secondary structure as well as those of the single N‐glycosylation acceptor site. The cytoplasmic domain was shorter by 38 amino acids than those of rat and shark P0 and except for a high proportion of basic amino acids did not show any appreciable sequence homology. A single mRNA species of 2 kb was identified by northern blotting, which was expressed in brain tissue but not in liver. By in situ hybridization a selective labeling of myelinating glial cells in the trout CNS and PNS was revealed. The developmental appearance of the IP1 transcript closely coincided with a period of active myelin deposition in most regions of the trout brain.
α‐Amino‐3‐Hydroxy‐5‐Methyl‐4‐Isoxazolepropionate (AMPA) Receptors Mediate Excitotoxicity in the Oligodendroglial Lineage
Authors: Yoshioka Akira , Hardy Mattie , Younkin Donald P. , Grinspan Judith B. , Stern Janet L. , Pleasure David
Published: July 2010

Abstract: We demonstrate by reverse transcriptase‐polymerase chain reaction and Southern blotting that an immortalized rat oligodendroglial cell line (CG‐4) expresses the non‐N‐methyl‐d‐aspartate (non‐NMDA) glutamate receptor (GluR) genes GluR2–7, KA‐1, and KA‐2 and that nonimmortalized cells of the rat oligodendroglial lineage express the GluR1–3, GluR5–7, KA‐1, and KA‐2 genes. Lactic dehydrogenase release assays show that both immortalized and nonimmortalized cells of the oligodendroglial lineage are damaged by a 24‐h exposure to 500 µM kainate or 5 mMl‐glutamate, but not by a 24‐h exposure to up to 10 mMα‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA). Damage is prevented by the non‐NMDA GluR channel inhibitor 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione and is also averted if Ca2+ is removed from the culture medium. Cyclothiazide, which blocks desensitization of AMPA‐preferring GluRs, increases cytotoxicity of kainate as well as inducing toxicity of AMPA. We conclude that cells of the oligodendroglial lineage express a population of AMPA‐preferring and possibly also kainate‐preferring GluR channels that are capable of mediating Ca2+‐dependent excitotoxicity and that AMPA‐induced cytotoxicity is blocked by desensitization of AMPA‐preferring GluRs.
Chronic Activation of Inhibitory δ‐Opioid Receptors Cross‐Regulates the Stimulatory Adenylate Cyclase‐Coupled Prostaglandin E1 Receptor System in Neuroblastoma × Glioma (NG108‐15) Hybrid Cells
Authors: Ammer Hermann , Schulz Rüdiger
Published: July 2010

Abstract: The present article investigates chronic opioid regulation of the stimulatory adenylate cyclase‐coupled prostaglandin E1 (PGE1) receptor system in neuroblastoma × glioma (NG108‐15) hybrid cells. Persistent activation of δ‐opioid receptors by morphine (10 µmol/L; 3 days) substantially down‐regulates the number of PGE1 binding sites by ∼30%, without affecting their affinity. Radioligand binding studies performed in the presence of GTPγS (100 µmol/L) further revealed that the remaining PGE1 binding sites are still capable of interacting functionally with their associated stimulatory G proteins, Gs. On the postreceptor level, neither changes in the abundance nor in the intrinsic activity of the α subunit of Gs (Gsα) were found during the state of opioid dependence, as has been verified by western blot and S49 cyc− reconstitution experiments, respectively. Evaluation of the functional interaction between PGE1 receptors and Gs by means of receptor‐stimulated, cholera toxin‐catalyzed ADP‐ribosylation of Gsα revealed a significant increase in the ability of PGE1 receptors to activate Gsα (3.3‐fold increase in EC50; p < 0.05) in cells chronically exposed to morphine. This effect was completely blocked by coincubation of the cells together with the opiate antagonist naloxone (100 µmol/L; 3 days), whereas precipitation of morphine withdrawal by naloxone (100 µmol/L) had no further effect on sensitization in PGE1 receptor/Gs coupling. These findings provide evidence that the stimulatory adenylate cyclase‐coupled PGE1 receptor system represents a potential target of chronic δ‐opioid receptor activation in NG108‐15 hybrid cells. They further suggest that sensitization in stimulatory signal transduction plays a critical role in the generation of opioid dependence.
Comparison of Dopamine Uptake in the Basolateral Amygdaloid Nucleus, Caudate‐Putamen, and Nucleus Accumbens of the Rat
Authors: Jones Sara R. , Garris Paul A. , Kilts Clinton D. , Wightman R. Mark
Published: July 2010

Abstract: Regional differences in the kinetics and pharmacological inhibition of dopamine uptake were investigated with fast‐scan cyclic voltammetry in both the intact rat brain and a brain slice preparation. The regions compared were the basolateral amygdaloid nucleus, caudate‐putamen, and nucleus accumbens. The frequency dependence of dopamine efflux evoked in vivo by electrical stimulation of the medial forebrain bundle was evaluated by nonlinear curve fitting with a Michaelis‐Menten‐based kinetic model. The Km for dopamine uptake was found to be significantly higher in the basolateral amygdala (0.6 µM) than in the other two regions (0.2 µM), whereas the Vmax value for dopamine uptake in the basolateral amygdala was significantly lower (0.49 µM/s vs. 3.8 and 2.4 µM/s in the caudate and accumbens, respectively). Similar kinetics were also obtained in brain slices. Addition of a dopamine uptake inhibitor, cocaine or nomifensine (10 µM), to the perfusion buffer increased the apparent Km value >25‐fold in slices of both the caudate‐putamen and nucleus accumbens. In contrast, neither uptake inhibitor had an observable effect in the basolateral amygdaloid nucleus. Thus, dopamine uptake in the rat brain is regionally distinct with regard to rate, affinity, and sensitivity to competitive inhibition.
Spontaneous Withdrawal from Long‐Term Treatment with Morphine Accelerates the Turnover of α2‐Adrenoceptors in the Rat Brain: Up‐Regulation of Receptors Associated with Increased Receptor Appearance
Authors: Gabilondo Ane M. , García‐Sevilla Jesús A.
Published: July 2010

Abstract: The aim of this study was to quantify and compare the turnover of brain α2‐adrenoceptors during chronic morphine treatment and after spontaneous morphine withdrawal in rats. The oral administration of increasing doses of morphine (10–90 mg/kg) for 20 days did not alter the specific binding of the agonist [3H]clonidine in the cerebral cortex. However, spontaneous opiate withdrawal (24 h) significantly increased the density of cortical α2‐adrenoceptors (Bmax for [3H]clonidine was 21% greater). The recovery of [3H]clonidine binding after irreversible inactivation by N‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (1.6 mg/kg) was assessed in naive, morphine‐dependent, and morphine‐withdrawn rats to study the process of α2‐adrenoceptor repopulation and to calculate receptor turnover parameters. The simultaneous analysis of receptor recovery curves revealed that the turnover of brain α2‐adrenoceptors in morphine‐withdrawn rats was accelerated [appearance rate constant (r) = 21 fmol/mg of protein/day; disappearance rate constant (k) = 0.25 day−1] compared with those in morphine‐dependent (r = 13 fmol/mg of protein/day; k = 0.14 day−1) and naive (r = 15 fmol/mg of protein/day; k = 0.16 day−1) rats. Moreover, this analysis also indicated that the increased density of cortical α2‐adrenoceptors observed during morphine withdrawal was due to a significantly higher receptor appearance (Δr = 37–57%) and not to a decreased receptor disappearance, which in fact showed also an increase (Δk = 56–79%). It is proposed that the increased rate of α2‐adrenoceptor production in the brain of morphine‐dependent rats during spontaneous withdrawal is most probably mediated by the overactivity of the adenylyl cyclase/cyclic AMP system induced by opiate addiction.
Alteration of Phospholipase C‐δ Protein Level and Specific Activity in Alzheimer's Disease
Authors: Shimohama Shun , Fujimoto Sadaki , Matsushima Hideyuki , Takenawa Tadafumi , Taniguchi Takashi , Perry George , Whitehouse Peter J. , Kimura Jun
Published: July 2010

Abstract: Phosphoinositide‐specific phospholipase C (PLC) is a key enzyme in signal transduction. We have previously demonstrated that an antibody to an isozyme of PLC, PLC‐δ, produced intense staining of neurofibrillary tangles in the brains of patients with Alzheimer's disease. In the present study, we investigated the protein level and activity of this enzyme in control and Alzheimer brains. Western blot analysis using a specific antibody for PLC‐δ showed that the concentration of PLC‐δ protein was significantly higher in the cytosolic fraction of Alzheimer's disease cortical tissue than in control brains. The activity of PLC‐δ, which hydrolyzes phosphatidylinositol, was also investigated, and we found that PLC‐δ activity was not significantly different in the Alzheimer and control cytosolic fractions. These results indicate that the specific activity of PLC‐δ is decreased in Alzheimer brains and suggest that inactivation of PLC‐δ might be related to the pathophysiology of this disease.
Multisite Interactions Between Pb2+ and Protein Kinase C and Its Role in Norepinephrine Release from Bovine Adrenal Chromaffin Cells
Authors: Tomsig Jose L. , Suszkiw Janusz B.
Published: July 2010

Abstract: We investigated the interaction between Pb2+ and protein kinase C (PKC) in the Pb2+‐induced release of norepinephrine (NE) from permeabilized adrenal chromaffin cells. Our analysis of endogenous PKC activity in permeabilized cells suggests that Pb2+ interacts with the adrenal enzyme at multiple sites. Pb2+ activates the enzyme through high‐affinity (KA(Pb) = 2.4 × 10−12M) interactions and inhibits the enzyme by competitive and noncompetitive interactions with nanomolar‐(Ki = 7.1 × 10−9M) and micromolar‐ (K′i = 2.8 × 10−7M) affinity sites, respectively. Activation of PKC by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) in Ca2+‐deficient, Pb2+‐containing medium, enhances the Pb2+‐induced NE release from permeabilized chromaffin cells by lowering the concentration of Pb2+ required for half‐maximal activation of the secretory response from 7.5 × 10−10 to 5.7 × 10−11M. The PKC inhibitors staurosporine and pseudosubstrate PKC (19–36) abolish the effect of TPA without affecting the Pb2+‐induced secretion in the absence of TPA. These results indicate that (a) Pb2+ is a partial agonist of PKC, capable of both activating and inhibiting the enzyme and (b) synergistic activation of PKC by TPA and Pb2+ results in increased sensitivity of exocytosis to Pb2+ but is not obligatory for Pb2+‐triggered secretion.
The 3′ Flanking Region of the Human Tyrosine Hydroxylase Gene Directs Reporter Gene Expression in Peripheral Neuroendocrine Tissues
Authors: Wong Shou C. , Moffat Mark A. , Coker George T. , Merlie John P. , O'Malley Karen L.
Published: July 2010

Abstract: Cell type‐specific expression of the catecholamine synthetic enzyme, tyrosine hydroxylase (TH), appears to be mediated in part by cis‐acting elements located at the 3′ end of the human gene. Further delineation of this region indicated sequences corresponding to a CACGTG motif significantly stimulated transcription of a heterologous promoter in various cell types. Mutation of this site led to a complete loss of activity. DNase footprinting, gel retardation, and UV cross‐linking experiments indicated that a 74‐kDa cellular factor(s) bound specifically to the CACGTG motif in the pheochromocytoma cell line PC12. The size of this protein and its pattern of expression are compatible with those of the CACGTG binding protein TFE3. Transgenic animals were created using a 261‐bp human TH 3′ fragment encompassing the CACGTG motif in front of a thymidine kinase promoter/chloramphenicol acetyltransferase reporter gene. In three lines of mice this fragment was sufficient to direct a pattern of mRNA expression in peripheral neuroendocrine tissues that mimicked TH mRNA distribution. However, these sequences were not sufficient for CNS‐specific patterns of expression. Thus, multiple cell type‐specific enhancers may regulate TH gene expression in the CNS and periphery.
All‐trans‐ and 9‐cis‐Retinoic Acid Enhance the Cholinergic Properties of a Murine Septal Cell Line: Evidence that the Effects Are Mediated by Activation of Retinoic Acid Receptor‐α
Authors: Pedersen Ward A. , Berse Brygida , Schüler Ulrike , Wainer Bruce H. , Blusztajn Jan Krzysztof
Published: July 2010

Abstract: We investigated the effects of retinoids on the cholinergic properties of a murine septal cell line, SN56. Treatment of the cells with all‐trans‐retinol (vitamin A), all‐trans‐retinal, all‐trans‐retinoic acid (t‐RA), 9‐cis‐retinoic acid (9c‐RA), or 13‐cis‐retinoic acid caused time‐ and concentration‐dependent increases in choline acetyltransferase activity (up to 3.4‐fold) and in intracellular acetylcholine levels (up to 2.5‐fold, with respective EC50 values of 68, 50, 18, 15, and 56 nM). Furthermore, treatment with either t‐RA or 9c‐RA at 1 µM for 48 h resulted in an increase in the expression of choline acetyltransferase mRNA by threefold that of controls. These data and the presence of putative retinoic acid response elements in the 5′ region of the murine choline acetyltransferase gene indicate that retinoids stimulate choline acetyltransferase transcription in murine cholinergic neurons. No additivity or synergism was observed between the effects of t‐RA and 9c‐RA on any of these cholinergic properties of SN56 cells, suggesting a common mechanism of action of the two retinoids. However, a combined treatment with t‐RA and forskolin, which activates adenylate cyclase, resulted in an additive increase in acetylcholine content. Using an antagonist selective for the retinoic acid receptor‐α subtype, Ro 41‐5253, we found that the effects of t‐RA and 9c‐RA on acetylcholine levels were abolished. An agonist selective for retinoic acid receptor‐α, Ro 40‐6055, increased acetylcholine levels to a similar extent as t‐RA and 9c‐RA, and this effect was blocked by the antagonist. Our results suggest that retinoids modulate the cholinergic phenotype of septal neurons by activation of retinoic acid receptor‐α.
Receptor‐Mediated Desensitisation of Histamine H1 Receptor‐Stimulated Inositol Phosphate Production and Calcium Mobilisation in GT1‐7 Neuronal Cells Is Independent of Protein Kinase C
Authors: Zamani M. Reza , Dupere Jonathan R. B. , Bristow David R.
Published: July 2010

Abstract: GT1‐7 cells, a clonal line derived from specific tumours of gonadotropin‐releasing hormone‐secreting neurons from mouse hypothalamus, were used as a model system to investigate the cellular mechanisms underlying the histamine H1 receptor‐mediated desensitisation. GT1‐7 cells contain H1 receptors, acute stimulation of which leads to the desensitisation of histamine‐mediated calcium mobilisation and is manifest as a concurrent reduction in both the magnitude of the calcium transient and of the sustained phase. Acute pretreatment of the cells with the phorbol ester, phorbol 12‐myristate 13‐acetate, can also ablate the histamine‐stimulated calcium mobilisation. In addition, acute H1‐receptor stimulation and acute phorbol ester treatment result in the attenuation of histamine‐mediated inositol phosphate production. Receptor desensitisation resulting from acute stimulation with histamine is not affected by inhibiting protein kinase C (PKC) activity with Ro 31‐7549 or staurosporine. In contrast, the desensitisation of H1‐receptor responses induced by direct activation of protein kinase C is preventable by PKC inhibitors. Thus, these results imply that a PKC‐dependent mechanism and PKC‐independent mechanism are involved in the H1‐receptor desensitisation cascade in GT1‐7 cells and do not support the involvement of PKC in the receptor‐mediated desensitisation of H1 receptor‐stimulated calcium and inositol phosphate responses.
Characterization of NMDA Receptor Subunit‐Specific Antibodies: Distribution of NR2A and NR2B Receptor Subunits in Rat Brain and Ontogenic Profile in the Cerebellum
Authors: Wang Yue‐hua , Bosy Thomas Z. , Yasuda Robert P. , Grayson Dennis R. , Vicini Stefano , Pizzorusso Tommaso , Wolfe Barry B.
Published: July 2010

Abstract: Selective antisera for NMDA receptor subunits NR2A and NR2B have been developed. Each antiserum identifies a single band on an immunoblot at ∼175 kDa that appears to be the appropriate subunit of the NMDA receptor. Using these antisera the relative densities of the subunits in eight areas of adult rat brain have been determined. The NR2A subunit was found to be at its highest level in hippocampus and cerebral cortex, to be at intermediate levels in striatum, olfactory tubercle, midbrain, olfactory bulb, and cerebellum, and to be at lowest levels in the pons‐medulla. The NR2B subunit was found to be expressed at its highest levels in the olfactory tubercle, hippocampus, olfactory bulb, and cerebral cortex. Intermediate levels were expressed in striatum and midbrain, and low levels were detected in the pons‐medulla. No signal for NR2B was found in the cerebellum. These regional distributions were compared with that for [3H]MK‐801 binding sites. It was found that although the distribution of the NR2A subunit corresponds well with radioligand binding, the distribution of the NR2B subunit does not. The ontogenic profiles of NR2A and NR2B subunits in the rat cerebellum were also determined. Just following birth [postnatal day (P) 2] NR2A subunits are undetectable, whereas NR2B subunits are expressed at amounts easily measurable. Beginning at about P12 the levels of NR2A rise rapidly to reach adult levels by P22. At the same time (P12), levels of NR2B protein begin to decline rapidly to reach undetectable levels by 22 days after birth. The results suggest that NMDA receptors are likely to be composed of different subunits in different parts of the brain and that even in the same tissue the receptors are likely to show different properties at various times during development due to alterations in the subunit composition of the receptor.
Effect of Acute Fluoxetine Treatment on the Brain Serotonin Synthesis as Measured by the α‐Methyl‐l‐Tryptophan Autoradiographic Method
Authors: Tsuiki Ko , Yamamoto Y. Lucas , Diksic Mirko
Published: July 2010

Abstract: The effect of treatment with acute fluoxetine, a serotonin reuptake inhibitor, on the rate of serotonin synthesis in the rat brain was studied through autoradiography following intravenous administration of α‐methyl‐l‐[3H]tryptophan. The rate of serotonin synthesis in fluoxetine‐treated rats was compared with the rate measured in sham‐treated rats (saline injection). Results showed a significant increase in the rate of synthesis in the majority of cerebral structures examined. The greatest increase (given as a percentage of rates in control animals) in the rate of serotonin synthesis was observed in the substantia nigra compacta (344%), hippocampus‐CA3 (337%), dorsal hippocampus (283%), and caudate‐putamen (232%). Fluoxetine had a less significant effect on the rate of synthesis in the pineal body (44%). Data suggest that acute fluoxetine treatment (30 mg/kg, i.p.) enhances the rate of serotonin synthesis in all the structures of rat brain examined in this work.
Investigation into the Role of N‐Acetylaspartate in Cerebral Osmoregulation
Authors: Taylor Deanna L. , Davies Siân E. C. , Obrenovitch Tihomir P. , Doheny Mary H. , Patsalos Philip N. , Clark John B. , Symon Lindsay
Published: July 2010

Abstract: Marked abnormalities of the magnetic resonance intensity of N‐acetylaspartate (NAA) have been reported in patients with various neurological disorders, but the neurochemical consequences of these alterations are difficult to assess because the function of NAA remains speculative. The purpose of this study was to examine whether NAA plays a role in protecting neurons against osmotic stress. Intracerebral microdialysis was used to expose a small region of the rat dorsolateral striatum to an increasingly hyposmotic environment and to measure resulting changes in NAA extracellular concentrations. NAA changes in the extracellular fluid (ECF) were compared with those of the amino acids, in particular, taurine, known to be involved in brain osmoregulation. Stepped increases in cellular hydration produced by hyposmotic perfusion media induced a marked increase in ECF NAA, reflecting a redistribution of NAA from intra‐to extracellular space. Parallel experiments showed that, of all the extracellular amino acids measured, only taurine markedly increased with hyposmolar perfusion medium, indicating that the ECF NAA increase associated with hyposmotic stress was a specific response and not passive leakage out of the cells. As NAA is predominantly neuronal, it may contribute to the protection of neurons against swelling (i.e., regulatory volume decrease). In conditions with impaired blood‐brain barrier and cytotoxic oedema, efflux of intracellular NAA subsequent to sustained cellular swelling might lead to a reduction in total brain NAA detectable by magnetic resonance spectroscopy. Alternatively, redistribution of NAA from intra‐to extracellular space implies changes in its chemical environment that may alter its magnetic resonance visibility.
Characterization of cDNA and Genomic Clones Encoding Human Myelin Oligodendrocyte Glycoprotein
Authors: Hilton Adrienne A. , Slavin Anthony J. , Hilton Douglas J. , Bernard Claude C. A.
Published: July 2010

Abstract: Myelin oligodendrocyte glycoprotein (MOG) is a transmembrane protein expressed only in the CNS and is a possible target autoantigen in multiple sclerosis (MS). To further study the association of MOG with MS, we have characterized cDNA and genomic clones encoding human MOG. The human MOG cDNA, like its rodent and bovine counterparts, encodes a mature protein containing an Ig‐like domain, followed by two potential membrane‐spanning regions. The intron‐exon boundaries of the human MOG gene were mapped and revealed that the signal peptide is encoded by the first exon, the Ig‐like domain of MOG is encoded on the second exon, whereas the remainder of the molecule is encoded by six shorter exons. In addition to the major cDNA species, a second class of MOG cDNA was isolated in which an intron was retained. Not only did this second cDNA species represent 30% of the clones analyzed (nine of 30), but RNA encoding this form was detectable by northern and reverse transcription‐polymerase chain reaction analysis of the brain and spinal cord. Furthermore, we describe several restriction fragment length polymorphisms of the human MOG gene, one of which may be associated with MS susceptibility.
Sympathetic Sprouting: Time Course of Changes of Noradrenergic, Cholinergic, and Serotonergic Markers in the Denervated Rat Hippocampus
Authors: Jackisch R. , Neufang B. , Hertting G. , Jeltsch H. , Kelche C. , Will B. , Cassel J. C.
Published: July 2010

Abstract: As a first step for experiments investigating the presynaptic characteristics of sympathetic fibers grown into the denervated hippocampus, we studied the time course of changes of neurochemical markers in the rat hippocampus, subsequent to aspiration lesions of the fimbria‐fornix and the overlying callosal and cortical structures. At various postsurgical delays (1, 2, 8, 24, and 40 weeks), the activity of choline acetyltransferase, the high‐affinity synaptosomal uptake of choline and noradrenaline, and the concentrations of noradrenaline, serotonin, and 5‐hydroxyindoleacetic acid were measured in a dorsal, an intermediate, and a ventral part of the hippocampus. Levels of all markers were significantly reduced shortly (1–2 weeks) after the lesions. However, whereas the cholinergic (choline uptake and choline acetyltransferase activity) and the serotonergic (concentrations of serotonin and 5‐hydroxyindoleacetic acid) markers remained significantly reduced for up to 40 weeks, both noradrenergic markers recovered to near‐normal (noradrenaline uptake) or even supranormal (noradrenaline concentration) levels, although with clear‐cut differences in the time course and the regional characteristics. The noradrenaline content reached control levels already 8 weeks after lesion surgery and was about two to three times higher 40 weeks later, with the most dramatic effects in the ventral hippocampus. In contrast, high‐affinity noradrenaline uptake reached control values only 24 weeks after lesion and exceeded them only in the ventral hippocampus 40 weeks after surgery. It is concluded (a) that hippocampal noradrenaline concentration is a more sensitive marker for sympathetic sprouting than high‐affinity noradrenaline uptake and (b) that functional in vitro studies on hippocampal sympathetic ingrowth appear to fit optimal conditions in the ventral hippocampus at a delay of at least 40 weeks after surgery.
Down‐Regulation of c‐neu Receptors by Nerve Growth Factor in PC12 Cells
Authors: Oshima Mari , Weiss Linnea , Dougall William C. , Greene Mark I. , Guroff Gordon
Published: July 2010

Abstract: A small number of p185c‐neu receptors have been found on PC12 cells. These receptors show some basal phosphorylation in quiescent cells. When the cells are treated with nerve growth factor (NGF) for a short time, some increase in phosphorylation is seen, mainly on serine and threonine residues, and this is accompanied by a slight shift in the apparent molecular weight. Epidermal growth factor (EGF) also increases the phosphorylation of p185c‐neu, in this case on tyrosine residues. Neither heregulin‐β1 nor gp30 stimulates the tyrosine phosphorylation of p185c‐neu, and neither has a proliferative effect on the cells. Treatment of the cells with NGF for 5 days produces a 70–80% reduction in the number of p185c‐neu receptors. This down‐regulation does not occur when PC12nnr5 cells, which lack the high‐affinity NGF receptor, p140trk, are treated with NGF.The level of p185c‐neu mRNA is not altered by NGF treatment, suggesting that the down‐regulation is due to either a translational or a posttranslational alteration.
Expression of Protein Kinase C Family Members in the Cerebral Endothelial Cells
Authors: Krizbai I. , Szabó G. , Deli M. , Maderspach K. , Lehel C. , Oláh Z. , Wolff J. R. , Joó F.
Published: July 2010

Abstract: The protein kinase C (PKC) family is composed of at least four conventional (α, βI, βII, and γ) and several related novel (δ, ε, η, and ζ) isoforms with different distribution and sensitivity to Ca2+ and phorbol esters. The enzyme is known to be present in cerebral endothelial cells. We have investigated the occurrence of seven isoforms (α, β, γ, δ, ε, η, and ζ) by using reverse transcriptase‐polymerase chain reaction in rat brain, in a freshly isolated brain microvessel fraction, in primary cultures of rat brain endothelial cells, in an immortalized rat brain endothelial cell line, and in aortic endothelial cell cultures. Brain tissue contained all seven investigated isoforms. A similar expression pattern was seen in freshly purified microvessels, but the PKC‐γ isoform could not be detected. Primary cultures of endothelial cells expressed PKC‐α, ‐β, ‐δ, ‐η, and ‐ε isoenzymes, whereas the immortalized cell line expressed PKC‐α, ‐δ, ‐ε, and ‐η. The rat aortic endothelium contained only PKC‐α and ‐δ isoforms. The variety of expression patterns of PKC family members in endothelial cells of different type may reflect differences in the functional responsiveness to environmental stimuli. Because PKC has been shown to be involved in the regulation of the blood‐brain barrier permeability, the presence of different isoforms may confer a sophisticated intracellular regulatory mechanism to the brain endothelial cells.
Tumor Necrosis Factor Receptors in Neuroblastoma SKNBE Cells and Their Regulation by Retinoic Acid
Authors: Chambaut‐Guérin Anne‐Marie , Martinez Marie‐Chantal , Hamimi Chafika , Gauthereau Xavier , Nunez Jacques
Published: July 2010

Abstract: The human neuroblastoma cell line SKNBE can be differentiated either by serum removal or by adding to the culture medium different morphogens, for instance, retinoic acid (RA), cyclic AMP derivatives, and phorbol esters. Both the differentiated and undifferentiated cells express the two types of membrane tumor necrosis factor (TNF) receptors (TNFRs) of 55 and 75 kDa (p55 and p75 TNFR, respectively) and also their soluble forms. After RA addition the number of the surface TNFRs per cell is increased approximately twofold, but the kinetics of expression are different, depending on the receptor type. The level of the mRNAs of 2.4 and 4.2 kb, which, respectively, encode the p55 and p75 TNFRs, is also increased during the time course of differentiation, and the kinetics of their expression are biphasic. In contrast, the number of TNFRs and the level of their encoding mRNAs remain unchanged after exposure of the cells to both a phorbol and a cyclic AMP derivative.
Ciliary Neurotrophic Factor Expression in Schwann Cells Is Induced by Axonal Contact
Authors: Lee Deborah A. , Zurawel Russell H. , Windebank Anthony J.
Published: July 2010

Abstract: In the PNS, ciliary neurotrophic factor (CNTF) is found in significant amounts in the adult sciatic nerve, localized to myelin‐related Schwann cells (SCs). Levels are undetectable in newborn but high in adult animals. After crush injury, CNTF production is reduced, recovering only as the nerve regenerates; if regeneration does not occur, CNTF levels remain low. We have examined the coupling of CNTF expression to myelination in vitro and in vivo to determine if axon‐SC contact without myelination is sufficient to induce CNTF expression. Embryonic day 15 (E15) rat dorsal root ganglion (DRG) neuron and SCs were cocultured. CNTF was not detected in the DRGs either at E15 or after 2 days in vitro (div) by western blotting. However, after 10 div, CNTF could be identified and remained at constant levels up to 30 div. Depriving the cultures of ascorbic acid prevents myelination but not axon‐SC contact. This did not affect CNTF protein production. Using reverse transcriptase‐linked PCR (RT‐PCR) techniques, no CNTF message was present in DRG from day 14 or 15 fetal rats; but by 6 days in culture, message was detected in both myelinating and nonmyelinating cultures. Isolated SC cultures, without axonal contact, failed to express CNTF protein; however, mRNA was detected by RT‐PCR. Embryonic SC can be induced to synthesize CNTF in culture by axonal contact. Active myelination is not required.
Cyclic GMP‐Dependent Protein Kinase Substrates in Rat Brain
Authors: Wang Xin , Robinson Phillip J.
Published: July 2010

Abstract: Cyclic GMP (cGMP)‐dependent protein kinase (PKG) has a limited substrate specificity, and only cerebellar G‐substrate has been demonstrated in brain. In view of the physiological importance of cGMP and PKG in the nervous system, it is important to identify endogenous PKG substrates in rat brain. We devised a combination of ion‐exchange and hydrophobic chromatographies to identify potential PKG substrates. Extracts from cytosol, peripheral membrane proteins, or a fraction enriched in Ca2+‐sensitive lipid‐binding proteins were partly purified and phosphorylated with purified PKG. Using whole extracts only a single specific PKG substrate—P34—was found. However, after chromatography we detected >40 distinct proteins that were phosphorylated by PKG to a much greater extent than by cyclic AMP‐dependent protein kinase or protein kinase C. Four PKG substrates—P140, P65, P32, and P18—were detected in the cytosol. Six PKG substrates—P130, P85 (doublet), P58, P54, and P38—were enriched from the Ca2+‐sensitive lipid‐binding protein fraction. In peripheral membrane fractions >30 relatively specific PKG substrates were enriched after chromatography, especially P130, P94, P58, P52, P45, P40, P36, P34, P28, P26, P24, and P20. These results indicate that brain is not lacking in PKG substrates and show that many are apparently quite specific substrates for this enzyme. The identification of some of these novel PKG substrates will facilitate understanding the role of cGMP signaling in the brain.
Histamine‐Stimulated Inositol Phospholipid Metabolism in Bovine Adrenal Medullary Cells: A Kinetic Analysis
Authors: Bunn Stephen J. , Saunders Heather I. , Dunkley Peter R.
Published: July 2010

Abstract: Histamine stimulation of bovine adrenal medullary cells rapidly activated phospholipase C. [3H]Inositol 1,4,5‐trisphosphate [[3H]Ins(1,4,5)P3] levels were transiently increased (200% of basal values between 1 and 5 s) before declining to a new steady‐state level of ∼140% of basal values. [3H]Inositol 1,4‐bisphosphate [[3H]Ins(1,4)P2] content increased to a maximal and maintained level of 250% of basal values after 1 s, whereas levels of [3H]inositol 1,3,4‐trisphosphate [[3H]‐Ins(1,3,4)P3], [3H]inositol 1,3‐bisphosphate, and [3H]‐inositol 4‐monophosphate ([3H]Ins4P) increased more slowly. The rapid responses were not reduced by the removal of extracellular Ca2+, but they were no longer sustained over time. The turnover rates of selected inositol phosphate isomers have been estimated in the intact cell. [3H]Ins(1,4,5)P3 was rapidly metabolized (t1/2 of 11 s), whereas the other isomers were metabolized more slowly, with t1/2 values of 113, 133, 104, and 66 s for [3H]Ins(1,3,4)P3, [3H]Ins(1,4)P2, an unresolved mixture of [3H]inositol 1‐ and 3‐monophosphate ([3H]Ins1/3P), and [3H]Ins4P, respectively. The calculated turnover rate of [3H]Ins(1,4,5)P3 was sufficient to account for the turnover of the combination of both [3H]Ins(1,4)P2 and [3H]Ins(1,3,4)P3 but not that of [3H]Ins1/3P or [3H]Ins4P. These observations demonstrate that histamine stimulation of these cells results in a complex Ca2+‐dependent and ‐independent response that may involve the hydrolysis of inositol phospholipids in addition to phosphatidylinositol 4,5‐bisphosphate.
Neuroprotective Strategies in a Model of Chronic Glutamate‐Mediated Motor Neuron Toxicity
Authors: Rothstein Jeffrey D. , Kuncl Ralph W.
Published: July 2010

Abstract: A dramatic loss of glutamate transport has been observed in sporadic amyotrophic lateral sclerosis and has been postulated to contribute to the disease. Experimentally, this hypothesis was corroborated by mimicking the chronic loss of glutamate transport in post‐natal rat spinal cord organotypic cultures through the use of glutamate transport inhibitors. This system is characterized by a relatively selective slow loss of ventral horn motor neurons resulting from glutamate transport inhibition. In this study, spinal cord organotypic cultures were used to test various drugs to evaluate their neuroprotective properties against this slow glutamate‐mediated neurotoxicity. The most potent neuroprotectants were drugs that altered glutamate neurotransmission, including non‐NMDA receptor antagonists (GYKI‐52466, PD144216, and PD139977) and drugs that could block presynaptic release or synthesis (riluzole and gabapentin). In addition, some antioxidants (U83836E and N‐t‐butyl‐α‐phenylnitrone) and inhibitors of nitric oxide synthesis (NG‐monomethyl‐l‐arginine acetate) were modestly neuroprotective. The calcium endonuclease inhibitor aurintricarboxylic acid and the calcium release inhibitor dantrolene also provided partial motor neuron protection. However, several antioxidants and calcium channel antagonists had no excitotoxic neuroprotectant activity. This system provides a preclinical screening method for the burgeoning number of drugs postulated for clinical trials in motor neuron disease and a model to evaluate the mechanisms of chronic glutamate toxicity.
Expression of the Chondroitin Sulfate Proteoglycans of Amyloid Precursor (Appican) and Amyloid Precursor‐Like Protein 2
Authors: Pangalos Menelas N. , Shioi Junichi , Robakis Nikolaos K.
Published: July 2010

Abstract: The Alzheimer amyloid precursor (APP) protein is a member of a family of glycoproteins that includes the amyloid precursor‐like proteins (APLPs). Previously, we showed that in C6 glioma cell cultures, secreted APP nexin II occurs as the core protein of a chondroitin sulfate proteoglycan (CSPG). Here, we report that among seven untransfected cell lines, expression of secreted APP CSPG was restricted to two cell lines of neural origin, namely, C6 glioma and Neuro‐2a neuroblastoma (N2a) cells. Addition of dibutyryl cyclic AMP in N2a cultures, a treatment that induces the neuronal phenotype in these cells, resulted in a significant reduction in the amount of the secreted APP CSPG, although secretion of APP was only marginally affected. Growth in the presence of serum increased the size of the secreted APP CSPG, suggesting that the number and/or length of the chondroitin sulfate (CS) chains attached to the core APP varies with growth conditions. Extensive mapping with epitope‐specific anti‐bodies suggested that a CS chain is attached within or proximal to the Aβ sequence of APP. In contrast to the restricted expression of the APP CSPG, expression of secreted APLP2 CSPGs was observed in all cell lines examined. After chondroitinase treatment, two core proteins of ∼100 and 110 kDa were obtained that reacted with an APLP2‐specific antiserum, suggesting that non‐transfected cell lines contain at least two endogenous APLP2 CSPGs, probably derived by alternative splicing of the APLP2 KPI domain. The fraction of the APLP2 proteins in the CSPG form was dependent on the particular cell line examined. The proteoglycan nature of APP and APLP2 suggests that addition of the CS glycosaminoglycan chains is important for the implementation of the biological function of these proteins. However, the differential expression of these two proteoglycans suggests that their physiological roles and their possible involvement in Alzheimer's disease may differ.