bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein Kinase A Phosphorylation of the Proteasome: A Contribution to the α‐Secretase Pathway in Human Cells
Authors: Marambaud Philippe , Wilk Sherwin , Checler Frederic
Published: July 2010

Abstract: The β‐amyloid precursor protein undergoes a physiological cleavage by α‐secretase that leads to the release of a secreted C‐terminally truncated fragment called APPα and likely concomitantly reduces the formation of the amyloidogenic Aβ peptide. Here we demonstrate that APPα secretion is increased by the protein kinase A (PKA) effectors 8‐bromo cyclic AMP and forskolin in human embryonic kidney cells (HK293), and that this can be prevented by a proteasome inhibitor. Furthermore, we establish that PKA effectors but not protein kinase C agonists increase the chymotrypsin‐like activity and phosphorylation state of the proteasome in vitro and in vivo in HK293 cells. Altogether, this report demonstrates that the α‐secretase pathway is under the control of PKA in human cells and that the proteasome likely contributes, either directly or through yet unknown intermediates, to the PKA‐stimulated APPα secretion in human cells.
Glial Regulation of α7‐Type Nicotinic Acetylcholine Receptor Expression in Cultured Rat Cortical Neurons
Authors: Jensen Janice J. , Winzer‐Serhan Ursula H. , Leslie Frances M.
Published: July 2010

Abstract: Primary embryonic cortical cultures were used as an in vitro model to evaluate the influence of glia on developmental expression of α7‐type nicotinic acetylcholine receptors in rat brain. In cells cultured in serum‐containing medium without mitotic inhibitors, specific 125I‐α‐bungarotoxin binding to α7‐type nicotinic receptors was maximal 4–8 days after plating. Treatment with 5′‐fluorodeoxyuridine (80 µM) from 1 to 3 days in vitro significantly reduced glial proliferation and concomitantly increased 125I‐α‐bungarotoxin binding, whereas plating onto a glial bed layer decreased binding. There was no significant binding to pure glial cultures. Treatment‐induced changes in neuronal binding resulted from alterations in receptor density, with no change in affinity. 5′‐Fluorodeoxyuridine treatment also increased cellular expression of α7 receptor mRNA but had no effect on N‐[3H]methylscopolamine binding to muscarinic receptors. Glial conditioned medium decreased 125I‐α‐bungarotoxin binding in both control and 5′‐fluorodeoxyuridine‐treated cultures, suggesting the release of a soluble factor that inhibits α7‐type nicotinic receptor expression. An additional mechanism of glial regulation may involve removal of glutamate from the surrounding medium, as added glutamate (200 µM) increased 125I‐α‐bungarotoxin binding in astrocyte‐poor cultures but not in those that were astrocyte enriched. These results suggest that glia may serve a physiological role in regulating α7‐type nicotinic receptors in developing brain.
Inhibitors of Histamine Methylation in Brain Promote Formation of Imidazoleacetic Acid, Which Interacts with GABA Receptors
Authors: Prell George D. , Morrishow Albert M. , Duoyon Erwin , Lee Wah S.
Published: July 2010

Abstract: In brain, the precursor of imidazoleacetic acid (IAA), a GABAA agonist but a GABAC antagonist, is not known. In the periphery, IAA derives from oxidation of histamine. But in brain, histamine is thought to be metabolized solely by histamine methyltransferase (HMT), forming tele‐methylhistamine (t‐MH) and tele‐methylimidazoleacetic acid (t‐MIAA). We showed that [3H]histamine (intracerebroventricularly) could be converted to IAA in brains of rats, a process increased by inhibition of HMT. This demonstrated that brain can oxidize histamine and suggested that endogenous histamine might also be oxidized if HMT activity were reduced. We examined, in rat cerebral cortex, effects of the following HMT inhibitors (mg/kg i.p.): metoprine (10), tacrine (10), velnacrine (10, 30), and physostigmine (1, 2). Tacrine was a potent inhibitor (Ki∼ 22 nM). To measure histamine in tissue that contained HMT inhibitors, we developed a gas chromatography‐mass spectrometry method. After 2 h, all drugs reduced endogenous levels of t‐MH and t‐MIAA and increased levels of histamine and IAA. Our results show that inhibition of HMT promotes oxidation of histamine in brain, probably by shunting histamine to an alternative metabolic pathway. Formation of IAA provides a novel interaction between histaminergic and GABAergic systems in brain. Accumulation of IAA should be considered when inhibitors of HMT are used to probe brain histamine function.
A Novel Regulation of Expression of the α‐Subunit of the G Stimulatory Protein by Dopamine via D1 Dopamine Receptors
Authors: Sidhu Anita , Kimura Kazuhiro
Published: July 2010

Abstract: Exposure of human neuroblastoma SK‐N‐MC cells to 100 µM dopamine (DA) for 72 h caused 70% loss of immunodetectable membrane‐bound levels of the α‐subunit of Gs. The loss in Gsα was accompanied by reduced (64.3 ± 0.35% of control values) NaF‐mediated stimulation of adenylyl cyclase and was independent of accumulated cyclic AMP (cAMP) levels, because neither forskolin nor dibutyryl cAMP treatment of cells mimicked the DA‐induced effects. The reduction in Gsα content was manifest at the transcriptional level; Gsα mRNA levels were attenuated to 56.5 ± 10% of control values after a 24‐h treatment of cells with 100 µM DA. The concentration of DA required to produce the half‐maximal decrease of Gsα mRNA content was 20 nM, similar to the high‐affinity binding value (8.5 nM) of DA to D1 sites. Gsα mRNA levels were also attenuated (52 ± 3.5% of control values) by the D1‐selective agonist SKF R‐38393 but not by forskolin or dibutyryl cAMP. Attenuation of Gsα mRNA levels by agonists was blocked by the D1‐selective antagonist SCH 23390. Stimulation of adenylyl cyclase‐inhibitory DA receptors, which are coexpressed in these cells, failed to down‐regulate Gsα mRNA, indicating that regulation of Gsα mRNA expression occurs specifically through chronic stimulation of D1 DA receptors.
Avian Melatonin Synthesis: Photic and Circadian Regulation of Serotonin N‐Acetyltransferase mRNA in the Chicken Pineal Gland and Retina
Authors: Bernard Marianne , Iuvone P. Michael , Cassone Vincent M. , Roseboom Patrick H. , Coon Steven L. , Klein David C.
Published: July 2010

Abstract: The circadian rhythms in melatonin production in the chicken pineal gland and retina reflect changes in the activity of serotonin N‐acetyltransferase (arylalkylamine N‐acetyltransferase; AA‐NAT; EC 2.3.1.87). Here we determined that the chicken AA‐NAT mRNA is detectable in follicular pineal cells and retinal photoreceptors and that it exhibits a circadian rhythm, with peak levels at night. AA‐NAT mRNA was not detected in other tissues. The AA‐NAT mRNA rhythm in the pineal gland and retina persists in constant darkness (DD) and constant lighting (LL). The amplitude of the pineal mRNA rhythm is not decreased in LL. Light appears to influence the phase of the clock driving the rhythm in pineal AA‐NAT mRNA in two ways: The peak is delayed by ∼6 h in LL, and it is advanced by >4 h by a 6‐h light pulse late in subjective night in DD. Nocturnal AA‐NAT mRNA levels do not change during a 20‐min exposure to light, whereas this treatment dramatically decreases AA‐NAT activity. These observations suggest that the rhythmic changes in chicken pineal AA‐NAT activity reflect, at least in part, clock‐generated changes in mRNA levels. In contrast, changes in mRNA content are not involved in the rapid light‐induced decrease in AA‐NAT activity.
Neurochemical Changes in the Cerebral Cortex of Treated and Untreated Hydrocephalic Rat Pups Quantified with In Vitro 1H‐NMR Spectroscopy
Authors: Harris N. G. , Plant H. D. , Inglis B. A. , Briggs R. W. , Jones H. C.
Published: July 2010

Abstract: The pathophysiology of infantile hydrocephalus is poorly understood, and shunt treatment does not always lead to a normal neurological outcome. To investigate some of the neurochemical changes in infantile hydrocephalus and the response to shunt treatment, we have used high‐resolution 1H‐NMR spectroscopy to analyze extracts of cerebral cortex from H‐Tx rats, which have inherited hydrocephalus with an onset in late gestation. Hydrocephalic rats and rats with shunts placed at either 4 or 12 days after birth were studied at 21 days after birth, together with age‐matched control littermates. In hydrocephalic rats there was a 46–62% reduction in the following compounds: myo‐inositol, creatine, choline‐containing compounds, N‐acetyl aspartate, taurine, glutamine, glutamate, aspartate, and alanine. Phosphocreatine, glycine, GABA, and lactate were also reduced but not significantly. These changes are consistent with neuronal atrophy rather than ischemic damage. In hydrocephalic rats that received shunt treatment at 4 days, there were no significant reductions in any chemicals, indicating a normal complement of neurons. However, some compounds, particularly taurine, were elevated above control. After treatment at 12 days, N‐acetyl aspartate and aspartate remained significantly reduced, suggesting continued neuronal deficiency.
Generation of Reactive Oxygen Species by Tyrosine Hydroxylase: A Possible Contribution to the Degeneration of Dopaminergic Neurons?
Authors: Haavik Jan , Almås Bjørg , Flatmark Torgeir
Published: July 2010

Abstract: It has been suggested that idiopathic parkinsonism, characterized by a loss of dopaminergic neurons of the nigrostriatal pathway, is due to the intracellular generation of reactive oxygen species, generated by a nonenzymatic or enzymatic partial reduction of dioxygen. Based on in vitro studies of the iron‐containing monooxygenase tyrosine hydroxylase (TH), evidence is presented that this enzyme system may also contribute to such an oxidative stress. Thus, the purified and Fe2+‐reconstituted recombinant human enzyme shows a time‐ and temperature‐dependent partial uncoupling of the hydroxylation of l‐tyrosine with the natural cofactor (6R)‐tetrahydrobiopterin, resulting in the formation of H2O2. The degree of uncoupling of the hydroxylation reaction is significantly higher when certain substrate analogues, notably the 7‐substituted isomer (7‐tetrahydrobiopterin) of the natural cofactor, is used. In the presence of H2O2 and Fe2+, the addition of TH increases the production of the highly reactive •OH radical, probably via a Fenton type of reaction. It is not clear whether this in vitro reaction can mediate cellular injury in vivo. However, it is known that the distribution of TH in the central and peripheral nervous system often corresponds to that of the neuronal degeneration in idiopathic parkinsonism, a finding that is compatible with a pathogenetic effect of TH.
Brain‐Derived Neurotrophic Factor Increases the Stimulation‐Evoked Release of Glutamate and the Levels of Exocytosis‐Associated Proteins in Cultured Cortical Neurons from Embryonic Rats
Authors: Takei Nobuyuki , Sasaoka Kumi , Inoue Ko , Takahashi Masami , Endo Yasuhisa , Hatanaka Hiroshi
Published: July 2010

Abstract: Differentiation and survival of neurons induced by neurotrophins have been widely investigated, but little has been reported about the long‐term effect of brain‐derived neurotrophic factor (BDNF) on synaptic transmission. Among many steps of neurotransmission, one important step is regulated release of transmitters. Therefore, the release of glutamate and GABA from cortical neurons cultured for several days with or without BDNF was measured by an HPLC‐fluorescence method. Although BDNF had little effect on the basal release of glutamate, high K+‐evoked release was greatly increased by BDNF. BDNF also tended to increase evoked release of GABA. Recently, several proteins involved in the step of “regulated release” have been identified. Thus, the effect of BDNF on the levels of these proteins was then investigated. Neurons were cultivated with or without BDNF, collected, and electrophoresed for western blotting. BDNF increased levels of synaptotagmin, synaptobrevin, synaptophysin, and rab3A, which were known as vesicle protein. Levels of syntaxin, SNAP‐25, and β‐SNAP were also increased by BDNF. In addition, the numbers of cored and clear vesicles in nerve terminals or varicosities were also increased by BDNF. These results raise the possibility that BDNF increases regulated release of neurotransmitters through the up‐regulation of secretory mechanisms.
Activation of Protein Kinase C‐α and Translocation of the Myristoylated Alanine‐Rich C‐Kinase Substrate Correlate with Phorbol Ester‐Enhanced Noradrenaline Release from SH‐SY5Y Human Neuroblastoma Cells
Authors: Goodall Anna R. , Turner Neil A. , Walker John H. , Ball Stephen G. , Vaughan Peter F. T.
Published: July 2010

Abstract: The aim of this study was to investigate the mechanism by which short‐term pretreatment with the phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (TPA; 100 nM) enhances noradrenaline (NA) release from the human neuroblastoma cell line SH‐SY5Y. Subcellular fractionation and immunocytochemical studies demonstrated that an 8‐min TPA treatment caused translocation of the α‐subtype of protein kinase C (PKC) from the cytosol to the plasma membrane. In contrast, TPA altered the distribution of PKC‐ε from cytosolic and membrane‐associated to cytoskeleton‐ and membrane‐associated TPA had no effect on the cytosolic location of PKC‐ζ. Subcellular fractionation studies also showed that the myristoylated alanine‐rich C‐kinase substrate (MARCKS), a major neuronal PKC substrate that has been implicated in the mechanism of neurotransmitter release, translocated from membranes to cytosol in response to an 8‐min TPA treatment. Under these conditions the level of phosphorylation of MARCKS increased threefold. The ability of TPA to enhance NA release and to cause the translocation and phosphorylation of MARCKS was inhibited by the PKC inhibitor Ro 31‐8220 (10 µM). Selective down‐regulation of PKC subtypes by prolonged exposure to phorbol 12,13‐dibutyrate (100 nM) attenuated the TPA‐induced enhancement of NA release and the translocation of MARCKS over an interval similar to that of down‐regulation of PKC‐α (but not ‐ε or ‐ζ). Thus, we have demonstrated a strong correlation between the translocation of MARCKS and the enhancement of NA release from SH‐SY5Y cells due to the TPA‐induced activation of PKC‐α.
Sequence and Functional Analysis of Cloned Guinea Pig and Rat Serotonin 5‐HT1D Receptors: Common Pharmacological Features Within the 5‐HT1D Receptor Subfamily
Authors: Wurch Thierry , Palmier Christiane , Colpaert Francis C. , Pauwels Petrus J.
Published: July 2010

Abstract: This study was undertaken to investigate the pharmacology of cloned guinea pig and rat 5‐hydroxytryptamine (serotonin; 5‐HT)1D receptor sites. Guinea pig, rat, and mouse 5‐HT1D receptor genes were cloned, and their amino acid sequences were compared with those of the human, dog, and rabbit. The overall amino acid sequence identity between these 5‐HT1D receptors is high and varies between 86 and 99%. The sequence homology is slightly more divergent (13–27%) in the N‐terminal extracellular region of these 5‐HT1D receptors. Guinea pig and rat 5‐HT1D receptors, stably and separately expressed in rat C6 glial cells, are negatively coupled to cyclic AMP formation upon stimulation with agonists, as previously found for cloned human 5‐HT1D receptor sites. The cyclic AMP data show some common pharmacological features for the 5‐HT1D receptors of guinea pig, rat, and human: an almost similar rank order of potency for the investigated 5‐HT1D receptor agonists, stereoselectivity for the binding affinity and agonist potency of R(+)‐8‐hydroxy‐2‐(di‐n‐propylamino)tetralin, and equal 5‐HT1D receptor‐mediated antagonist potency for methiothepin and the 5‐HT2 receptor antagonists ritanserin and ketanserin. In conclusion, the pharmacology of the cloned 5‐HT1D receptor subtype seems, unlike the 5‐HT1B receptor subtype, conserved among various mammal species such as the human, guinea pig, and rat.
l‐(−)‐Desmethylselegiline, a Metabolite of Selegiline [l‐(−)‐Deprenyl], Protects Mesencephalic Dopamine Neurons from Excitotoxicity In Vitro
Authors: Mytilineou Catherine , Radcliffe Pheona M. , Olanow C. Warren
Published: July 2010

Abstract: Selegiline [l‐(−)‐deprenyl], a monoamine oxidase B inhibitor, has been used in the treatment of Parkinson's disease as a putative neuroprotective agent. Selegiline is metabolized rapidly in the gastrointestinal tract and liver to desmethylselegiline (DMS) and methamphetamine. We have previously shown that selegiline protects dopamine neurons in mesencephalic cultures from toxicity resulting from activation of glutamate receptors. In the present study we examined whether DMS has similar neuroprotective effects. Our data show that DMS protects dopamine neurons from N‐methyl‐d‐aspartate receptor‐mediated excitotoxic damage. The efficacy of DMS is greater than that of selegiline, as it can cause protection at lower concentrations and provide significantly greater levels of protection at the same concentrations. Our results suggest that DMS might be the active compound responsible for the neuroprotective properties of selegiline.
Cyclic GMP‐Dependent Protein Kinase and Cellular Signaling in the Nervous System
Authors: Wang Xin , Robinson Phillip J.
Published: July 2010

Abstract: Nitric oxide (NO) and natriuretic peptide hormones play key roles in a surprising number of neuronal functions, including learning and memory. Most data suggest that they exert converging actions by elevation of intracellular cyclic GMP (cGMP) levels through activation of soluble and particulate guanylyl cyclases. However, cGMP is only the starting point for multiple signaling cascades, which are now beginning to be defined. A primary action of elevated cGMP levels is the stimulation of cGMP‐dependent protein kinase (PKG), the major intracellular receptor protein for cGMP, which phosphorylates substrate proteins to exert its actions. It has become increasingly clear that PKG mediates some of the neuronal effects of cGMP, but how is not yet clear. One clear illustration of this pathway has been reported in striatonigral nerve terminals, where NO mediates phosphorylation of the protein phosphatase regulator dopamine‐ and cyclic AMP‐regulated phosphoprotein having a molecular mass of 32,000 (DARPP‐32) by PKG. There are remarkably few PKG substrates in brain whose identities are known. A survey of these proteins and those known from other tissues that might also be found in the nervous system reveals the key molecular sites where cGMP and PKG signaling is likely to be regulating neural function. These potential substrates are critically placed to have profound effects on the protein phosphorylation network through regulation of protein phosphatases, intracellular calcium levels, and the function of many ion channels and neurotransmitter receptors. The brain also contains a rich diversity of specific PKG substrates whose identities are not yet known. Their future identification will provide exciting new leads that will permit better understanding of the role of PKG signaling in both basic and higher orders of brain function.
Biochemical Evidence for the Existence of a Pool of Unassembled C2 Exon‐Containing NR1 Subunits of the Mammalian Forebrain NMDA Receptor
Authors: Chazot Paul L. , Stephenson F. Anne
Published: July 2010

Abstract: Optimum conditions were determined for the solubilisation of native NMDA receptors of adult mammalian brain with the retention of [3H]MK‐801 radioligand binding activity. The most efficient conditions were 1% Triton X‐100/1 M NaCl. The efficiency of solubilisation was as follows: cloned NMDA receptors expressed in mammalian cells > forebrain receptors > cerebellar receptors. Triton X‐100/1 M NaCl‐solubilised forebrain NMDA receptors had a molecular size of 710,000 daltons, but significant NR1 immunoreactivity (41%) migrated as a monomer of 125,000 daltons. Immunoaffinity purification of NMDA receptors from forebrain by anti‐NR1 911–920 antibody affinity chromatography from 1% Triton X‐100/1 M NaCl solubilised extracts yielded purification of the NR1 Mr 120,000 immunoreactive species, but no detectable NR2A or NR2B immunoreactivity. Immunoprecipitation of NMDA receptors from Triton X‐100/1 M NaCl extracts with anti‐NR1 911–920 antibodies also resulted in precipitation of NR1 subunits, but with no detectable NR2A or NR2B subunits. In contrast, by immunoprecipitation with anti‐NR1 17–35 antibodies, which recognise all forms of NR1, NR1, NR2A, and NR2B immunoreactivities were detected in the immune pellets. Similarly, a coassociation of NR1, NR2A, and NR2B subunits was demonstrated following extraction of forebrain membranes with 1% sodium deoxycholate (pH 9) and purification by anti‐NR1 911–920 antibody affinity chromatography. These results are consistent with the identification of a pool of unassembled C2 exon‐containing NR1 subunits, i.e., NR1‐1a, NR1‐1b, NR1‐2a, and NR1‐2b, selectively solubilised by 1% Triton X‐100/1 M NaCl.
Identification of Ser‐Pro and Thr‐Pro Phosphorylation Sites in Chicken Neurofilament‐M Tail Domain
Authors: Bennett Gudrun S. , Quintana Ricardo
Published: July 2010

Abstract: The tail domain of the midsize chicken neurofilament polypeptide (NF‐M) contains several different types of Ser‐Pro and Thr‐Pro putative phosphorylation sites. We determined which of these sites are actually phosphorylated in vivo. Chick sensory neuron cultures were incubated in [32P]phosphate, and the cytoskeletal fraction was mixed with a neurofilament fraction prepared from adult chicken brain. NF‐M was purified by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and digested with chymotrypsin, and two large fragments were isolated. These were individually cleaved with trypsin, endoprotease Lys‐C, or endoprotease Glu‐C, and peptides separated by two‐dimensional high‐voltage electrophoresis and thin‐layer chromatography. 32P‐labeled phosphopeptides were eluted from the cellulose plates and subjected to microsequencing and mass spectometry. We found that of 21 potential Ser‐Pro and Thr‐Pro phosphoacceptor sites, at least 20 are phosphorylated in vivo: all four Lys‐Ser‐Pro sites and at least 16 of the 17 Lys‐Xaa‐Xaa‐Ser/Thr‐Pro repeats. In addition, a novel Ser‐Pro site in the extreme carboxy terminus is phosphorylated. This site, which has no proximal Lys residue, is also found in mammalian NF‐M, but has not been reported to be phosphorylated. Together with three casein kinase I sites we have found recently in the acidic amino‐terminal segment of the tail, a total of 24 or 25 Ser and Thr phosphoacceptor sites have now been located in the chicken NF‐M tail.
The Low‐Density Lipoprotein Receptor‐Related Protein, a Multifunctional Apolipoprotein E Receptor, Modulates Hippocampal Neurite Development
Authors: Narita Masaaki , Bu Guojun , Holtzman David M. , Schwartz Alan L.
Published: July 2010

Abstract: The ε4 allele of apolipoprotein E (apoE) is an important risk factor for Alzheimer's disease. A major neuronal receptor for apoE within the brain is the low‐density lipoprotein receptor‐related protein (LRP). Using primary cultured hippocampal neurons, we examined the role of LRP in early neuronal development. LRP, as well as a 39‐kDa protein that regulates its activity, is localized abundantly in developing neurons. Both the 39‐kDa protein and an anti‐LRP antibody inhibited neurite outgrowth of primary hippocampal neurons cultured in either serum‐containing medium or on cortical astrocyte monolayers in serum‐free medium. It is noteworthy that microtubule‐associated protein‐2 immunoreactive process outgrowth was decreased significantly in hippocampal neurons cultured on cortical astrocytes derived from apoE‐deficient mice and was not diminished further following incubation with LRP inhibitors. Thus, these results suggest that LRP can influence aspects of neuronal process development and that apoE‐containing lipoproteins may be one of the major LRP ligands that can contribute to this process.
Nonmuscle Myosin Heavy Chain B Is Recognized by a Monoclonal Antibody that Inhibits GM1‐Enhanced Neuritogenesis
Authors: Mummert Christine M. , Schengrund Cara‐Lynne
Published: July 2010

Abstract: Addition of gangliosides to neuroblastoma cells maintained in vitro has been shown to enhance neuritogenesis. Although the mechanism by which they exert this effect is unknown, it has been postulated that they may act by adhering to cell surface proteins. In this article, we describe the isolation and identification of an S20Y murine neuroblastoma cell protein recognized by a monoclonal antibody that was prepared against putative GM1‐binding proteins and shown to inhibit GM1‐enhanced neuritogenesis. The protein identified was nonmuscle myosin heavy chain B, which appears to function in neurite formation but may not adhere to gangliosides.
Residues Specifically Involved in Down‐Regulation but Not Internalization of the m1 Muscarinic Acetylcholine Receptor
Authors: Shockley Melinda S. , Burford Neil T. , Sadée Wolfgang , Lameh Jelveh
Published: July 2010

Abstract: Human m1 muscarinic acetylcholine receptor mutants were screened to determine receptor domains and cellular pathways relevant to down‐regulation. Mutations in the second intracellular loop and the junctions of the third intracellular loop of the receptor, where a role for receptor activation or internalization had been previously demonstrated in HEK293 cells, were selected for this study. To assess receptor down‐regulation, the m1 receptor mutants were transfected into Chinese hamster ovary cells. Because receptor internalization is expected to precede down‐regulation, mutants displaying intact internalization were selected to permit interpretation of mutational effects on down‐regulation alone. Four mutations were identified that specifically impaired down‐regulation without altering receptor internalization: V127A, I211A, E360A, and K362A. The results define new receptor domains in the second intracellular loop and the junctions of the third intracellular loop that are involved in down‐regulation. These same four mutants were also defective in signaling via the phospholipase C and the adenylyl cyclase pathways and in G protein activation, as measured by [35S]GTPγS binding. However, the level of second messenger stimulation correlated poorly with the extent of down‐regulation. In summary, several mutations of the m1 receptor selectively affect down‐regulation, demonstrating that internalization and down‐regulation represent distinct events driven by different cellular mechanisms.
Evidence that GABAA Receptor Subunit mRNA Expression During Development Is Regulated by GABAA Receptor Stimulation
Authors: Poulter Michael O. , Ohannesian Lillith , Larmet Yves , Feltz Paul
Published: July 2010

Abstract: The expression of six mRNA species (α2, α3, α5, β2, β3, and γ2) encoding for GABAA receptor subunits was followed in cultured early postnatal cortical neurons by in situ hybridization histochemistry. In untreated control cultures it was found that these subunit mRNA expression profiles closely follow those seen during development in vivo. α3, α5, and β3 subunit expression declined, α2 expression increased, whereas β2 and γ2 subunit mRNA expression remained relatively constant. To test the hypothesis that GABAA receptor stimulation regulates these expression profiles, we tested the effect of a GABAA receptor positive modulator, allopregnanolone, and a GABAA receptor noncompetitive antagonist, tert‐butylbicyclophosphorothionate (TBPS). It was found that allopregnanolone augmented the rate at which the α3, α5, or β3 subunit mRNA expression declined and prevented the increase in α2 subunit mRNA expression. As well, allopregnanolone down‐regulated β2 subunit mRNA expression. TBPS, on the other hand, up‐regulated α3, α5, β2, and β3 subunit mRNA expression. It also down‐regulated the expression of α2 subunit mRNA. Both allopregnanolone and TBPS had no effect on γ2 subunit mRNA expression. These results imply that the developmental switchover of GABA receptor subunit mRNA expression is regulated by GABAA receptor activity.
Norepinephrine Microinjections in the Hypothalamic Paraventricular Nucleus Increase Extracellular Dopamine and Decrease Acetylcholine in the Nucleus Accumbens: Relevance to Feeding Reinforcement
Authors: Hajnal András , Mark Gregory P. , Rada Pedro V. , Lénárd László , Hoebel Bartley G.
Published: July 2010

Abstract: Norepinephrine (NE) was microinjected into the paraventricular nucleus (PVN), while microdialysis was used to monitor extracellular dopamine (DA) and acetylcholine (ACh) in the nucleus accumbens (NAc). The PVN is a site where exogenously administered NE can act through α2 receptors to elicit eating behavior and preference for carbohydrates. It was hypothesized that NE in the PVN acts on a behavior reinforcement system by altering the DA/ACh balance in the NAc. NE microinjections (80 nmol in 0.3 µl), which effectively elicited feeding in satiated rats in a separate test, caused a significant increase in extracellular DA (109%) and decrease in ACh (−27%) when the same animals were tested in the absence of food. In contrast when the food was available and ingested, ACh increased (51%) instead of decreasing. These results support the hypothesis that a functional link exists between the PVN and the NAc in which DA helps initiate and ACh helps stop appetitive behavior involved in the reinforcement of eating.
Dopaminergic and Serotonergic Neurotransmission Systems Are Differentially Involved in Auditory Cortex Learning: A Long‐Term Microdialysis Study of Metabolites
Authors: Stark Holger , Scheich Henning
Published: July 2010

Abstract: Auditory cortex has been shown to be a site of widespread neuronal learning processes even in the context of simple auditory conditioning behavior. In view of their presumed role in determining behavioral and motivational relevance of incoming information we investigated whether the dopaminergic and serotonergic systems are involved in auditory cortex learning. Using a chronic brain microdialysis technique over 4 days, samples from auditory cortex were obtained before, during, and after daily footshock avoidance training simultaneously from trained gerbils and passive control animals or pseudotrained animals. Because of detection limits of dopamine and serotonin in auditory cortex, the response profiles of extracellular homovanillic acid as the metabolite of the dopaminergic system and of 5‐hydroxyindoleacetic acid as the metabolite of the serotonergic system were determined from consecutive dialysis samples each day. The response of the dopaminergic system appeared to reflect the initial formation of the behaviorally relevant association exclusively during the first training day, whereas the serotonergic response appeared to correlate with the stress level of animals.
Prion Protein Fragment 106–126 Differentially Induces Heme Oxygenase‐1 mRNA in Cultured Neurons and Astroglial Cells
Authors: Rizzardini M. , Chiesa R. , Angeretti N. , Lucca E. , Salmona M. , Forloni G. , Cantoni L.
Published: July 2010

Abstract: Heme oxygenase (HO), which catalyzes the degradation of heme, has two isozymes (HO‐1 and HO‐2). In brain the noninducible HO‐2 isoform is predominant, whereas the inducible HO‐1 is a marker of oxidative stress. Because brain oxidative stress might be present in prion‐related encephalopathies (PREs), as in other neurodegenerative diseases, we investigated whether HO‐1 mRNA was induced in neuronal and astroglial cell cultures by a peptide corresponding to residue 106–126 of human prion protein (PrP). This peptide is amyloidogenic, and when added in vitro to cultured cells it reproduces the neuronal death and astroglial proliferation and hypertrophy occurring in PREs. HO‐1 mRNA did not accumulate in rat cultured neurons from hippocampus or cortex exposed to PrP 106–126 (50 µM for 5 days). PrP 106–126 induced HO‐1 mRNA accumulation in rat astroglial cultures depending on the exposure time and concentration, being maximal (33‐fold) after 7 days of exposure at 50 µM. The nonamyloidogenic amidated or amidated‐acetylated PrP 106–126 was ineffective, as was a scrambled peptide used as control. N‐Acetylcysteine reduced (50%) the accumulation of HO‐1 mRNA in astroglial cells after PrP 106–126 (25 µM) given for 5 days. Thus, oxidative stress is apparently a feature of the toxicity of PrP 106–126, and it might also occur in PREs; induction of HO‐1 could contribute to the greater resistance of astrocytes compared with neurons to PrP 106–126 toxicity.
Melatonin Synthesis in the Bovine Pineal Gland Is Regulated by Type II Cyclic AMP‐Dependent Protein Kinase
Authors: Maronde E. , Middendorff R. , Telgmann R. , Müller D. , Hemmings B. , Taskén K. , Olcese J.
Published: July 2010

Abstract: We investigated the expression of regulatory (R) and catalytic (C) subunits of cyclic AMP‐dependent protein kinase (cAK; ATP:protein phosphotransferase; EC 2.7.1.37) in the bovine pineal gland. In total RNA extracts of bovine pineal glands moderate levels of RIα/RIIβ and high levels of Cα and Cβ mRNA were found. We were able to detect a strong signal for RII and C subunit at the protein level, whereas RI was apparently absent. Probing sections of the intact bovine pineal gland with RI and RII antibodies stained only RII in pinealocytes. Pairs of cyclic AMP analogues complementing each other in activation of type II cAK, but not cAKI‐directed analogue pairs, showed synergistic stimulation of melatonin synthesis. Moreover, melatonin synthesis stimulated by the physiological activator norepinephrine in pineal cell cultures was inhibited by cAK antagonists. Taken together these results show the presence of RII regulatory and both Cα and Cβ catalytic subunits and thus cAKII holoenzyme in the bovine pineal gland. The almost complete inhibition of norepinephrine‐mediated melatonin synthesis by the cAK antagonists emphasizes the dominant role of cyclic AMP as the second messenger and cAK as the transducer in bovine pineal signal transduction.
Epinephrine and Norepinephrine Act as Potent Agonists at the Recombinant Human Dopamine D4 Receptor
Authors: Lanau Fabienne , Zenner Marie‐Thérèse , Civelli Olivier , Hartman Deborah S.
Published: July 2010

Abstract: The catecholamines dopamine (DA), epinephrine (EP), and norepinephrine (NE) play important roles in learning and memory, emotional states, and control of voluntary movement, as well as cardiovascular and kidney function. They activate distinct but overlapping neuronal pathways through five distinct DA receptors (D1R–D5R) and at least 10 different adrenergic receptors (α1a/b/c, α2a/b/c‐1/c‐2, and β1/β2/β3). The D4R, which is localized to mesolimbic areas of the brain implicated in affective and emotional behavior, has a deduced amino acid sequence with homology to both adrenergic and dopaminergic receptor subtypes. We report here that DA, EP, and NE all show binding in the nanomolar range to three isoforms of the recombinant human D4R (hD4R): D4.2, D4.4, and D4.7. Submicromolar concentrations of DA, EP, and NE were sufficient to activate hD4R isoforms in two different functional assays: agonist‐induced guanosine 5′‐O‐(3‐[35S]thiotriphosphate) binding and modulation of adenylyl cyclase activity. DA was approximately fivefold more potent than EP and NE at the D4R, whereas activation of the human D2R required at least 100‐fold higher catecholamine concentrations. Functional activation of the D4R by multiple neurotransmitters may provide a novel mechanism for integration of catecholamine signaling in the brain and periphery.
Stably Transfected Human Cells Overexpressing Rat Brain Endopeptidase 3.4.24.16: Biochemical Characterization of the Activity and Expression of Soluble and Membrane‐Associated Counterparts
Authors: Vincent Bruno , Dauch Pascale , Vincent Jean Pierre , Checler Frédéric
Published: July 2010

Abstract: We recently cloned endopeptidase‐24.16 (neurolysin; EC 3.4.24.16), a neurotensin‐degrading peptidase likely involved in the physiological termination of the neurotensinergic signal in the central nervous system and in the gastrointestinal tract. We stably transfected human kidney cells with the pcDNA3‐λ7aB1 construction bearing the whole open reading frame encoding the rat brain peptidase. Transfectants displayed endopeptidase‐24.16 immunoreactivity and exhibited QFS‐ and neurotensin‐hydrolyzing activities, the biochemical and specificity properties of which fully matched those observed with the purified murine enzyme. Cryoprotection experiments and substrate degradation by intact plated cells indicated that transfectants exhibited a membrane‐associated form of endopeptidase‐24.16, the catalytic site of which clearly faced the extracellular domain. Transfected cells were unable to secrete the enzyme. Overall, our experiments indicate that we have obtained stably transfectant cells that overexpress an enzymatic activity displaying biochemical properties identical to those of purified endopeptidase‐24.16. The membrane‐associated counterpart and lack of secretion of the enzyme were clearly reminiscent of what was observed with pure cultured neurons, but not with astrocytes. Therefore, the transfected cell model described here could prove useful for establishing, by a mutagenesis approach, the structural elements responsible for the “neuronal” phenotype exhibited by the enzyme in transfected cells.
Inhibition of Bradykinin‐Induced Calcium Increase by Phosphatase Inhibitors in Neuroblastoma × Glioma Hybrid NG108‐15 Cells
Authors: Hu Shei‐Ying , Song Shu‐Ling , Ho Mei‐Ying , Chueh Sheau‐Huei
Published: July 2010

Abstract: Prior treatment of NG108‐15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca2+ concentration ([Ca2+]i) induced by bradykinin by ∼63%. This inhibition was dependent on the concentration of okadaic acid with an IC50 of 0.15 nM. Okadaic acid treatment only lowered the maximal response of [Ca2+]i increase and had no effect on the EC50 value for bradykinin regardless of the presence of extracellular Ca2+. Neither the capacity of 45Ca2+ accumulation within intracellular nonmitochondrial Ca2+ stores nor the magnitude of [Ca2+]i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin‐evoked inositol 1,4,5‐trisphosphate (IP3) generation, the Mn2+ influx, and the capacity of mitochondrial Ca2+ accumulation. Furthermore, the sensitivity of IP3 in the Ca2+ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin‐induced [Ca2+]i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP3, and the slowed rate of Ca2+ influx. Thus, phosphorylation plays a role in bradykinin‐sensitive Ca2+ signaling cascade in NG108‐15 cells.
Distribution and Subcellular Localization of High‐Molecular‐Weight Microtubule‐Associated Protein‐2 Expressing Exon 8 in Brain and Spinal Cord
Authors: Shafit‐Zagardo Bridget , Kalcheva Nellie , Dickson Dennis , Davies Peter , Kress Yvonne
Published: July 2010

Abstract: The expression of high‐molecular‐weight (HMW) microtubule‐associated protein‐2 (MAP‐2) expressing exon 8 (MAP‐2+8) was examined by immunoblotting during rat brain development and in sections of human CNS. In rat brain, HMW MAP‐2+8 expression was detected at embryonic day 21 and increased during postnatal development. In adult rats, HMW MAP‐2+8 comigrated with MAP‐2a. In human adult brain, HMW MAP‐2+8 was expressed in select neuronal populations, including pyramidal neurons of layers III and V of the neocortex and parahippocampal cortex, pyramidal neurons in the endplate, CA2 and subiculum of the hippocampus, and the medium‐sized neurons of the basal ganglia. In the cerebellum, a subpopulation of Golgi neurons in the internal granular cell layer and most Purkinje cells were also stained. In the spinal cord staining was observed in large neurons of the anterior horn. Staining was present in cell bodies and dendrites but not in axons. At the ultra‐structural level, HMW MAP‐2+8 immunoreactivity was observed on mitochondrial membranes and in postsynaptic densities (PSDs) of some asymmetric synapses in the midfrontal cortex and spinal cord. Immunoblots of proteins isolated from enriched mitochondrial and PSD fractions from adult human frontal lobe and rat brains confirmed the presence of HMW MAP‐2+8. The presence of HMW MAP‐2+8 in dendrites and in close proximity to PSDs supports a role in structural and functional attributes of select excitatory CNS synapses.
Hypoxia Dramatically Increases the Nonspecific Transport of Blood‐Borne Proteins to the Brain
Authors: Plateel Monique , Teissier Elisabeth , Cecchelli Roméo
Published: July 2010

Abstract: Increased cerebrovascular permeability is an important factor for the development of cerebral edema. To investigate the effect of hypoxia on the transport of blood‐borne proteins to the brain, we used a cell culture model of the blood‐brain barrier (BBB) consisting of a coculture of brain capillary endothelial cells and astrocytes that closely mimics the in vivo situation. The permeability of albumin, a marker of the nonspecific transcellular route, is extremely low in this in vitro model of the BBB. After hypoxia, a huge increase in the permeability of albumin is detected. Despite the opening of the tight junctions already demonstrated after hypoxia, the increase in the permeability of albumin is mainly attributed to an increase of the nonspecific vesicular transport in the cell, attested by the temperature dependence of the phenomenon and the visualization of labeled apotransferrin in the cytoplasm. The increase of this pathway could participate in the development of brain edema during hypoxia.
Expression of Sulfated Gangliosides in the Central Nervous System
Authors: Farrer Robert G. , Quarles Richard H.
Published: July 2010

Abstract: Several sulfated lipids were detected in the ganglioside fraction isolated from a cell line of oligodendrocyte progenitors that had been metabolically labeled with [35S]sulfate. Separation of the ganglioside fraction by two‐dimensional TLC showed that, except for galactosylceramide‐sulfate, none of the sulfate‐labeled lipids comigrated with those glycosphingolipids visualized by orcinol staining, indicating that these sulfolipids were quantitatively minor components. At least eight sulfate‐labeled lipid bands were susceptible to desialylation by Arthrobacter ureafaciens neuraminidase, which resulted in the formation of three new bands that retained the labeled sulfate. Six of the sulfate‐labeled lipid bands containing sialic acid were also susceptible to Vibrio cholerae neuraminidase, which generated two labeled bands that appeared identical to the two major products formed after treatment with A. ureafaciens neuraminidase. In vivo labeling of lipids from 14‐day‐old rat brain with [35S]sulfate demonstrated that the synthesis of sulfated lipids containing sialic acid also occurred in intact brain tissue. These results show that sulfated gangliosides are synthesized in the CNS and that oligodendrocytes are one cell type that contributes to this synthesis.
Cloning of the Rat Fibroblast Growth Factor‐2 Promoter Region and Its Response to Mitogenic Stimuli in Glioma C6 Cells
Authors: Pasumarthi Kishore B. S. , Jin Yan , Cattini Peter A.
Published: July 2010

Abstract: Basic fibroblast growth factor (FGF‐2) is abundant in the developing and adult brain and has been linked with the origin and growth of neuronal and glial cells. Glial cells produce high levels of FGF‐2, stimulating autocrine growth as well as the survival and function of neurons in a paracrine manner. Abnormal levels of FGF‐2 have been linked with Alzheimer's, Huntington's, and Parkinson's diseases. Recent evidence has suggested that a component of the mitogenic response of glial cells is exerted on FGF‐2 at the transcriptional level. To assess transcriptional regulation of this potent growth factor, we cloned a 1.4‐kb genomic fragment containing the rat FGF‐2 promoter region. DNA blotting results indicated that the rat FGF‐2 gene exists as a single copy in the genome. The promoter region contains no TATA box but appears to rely instead on multiple GC‐rich start sites (P0, P1, and P2) for transcription initiation in rat brain as well as C6 glioma cells. One of these sites (P0) was located within four nucleotides of the reported 5′ end of the rat brain cDNA and constituted part of a consensus Egr‐1 binding site (5′‐GCGGGGGCG‐3′). Transcription from this site could be stimulated in C6 glioma cells in response to phorbol ester treatment. The induction of a “new” site (Pi) with phorbol ester also suggested a mechanism to explain the discrepancy between the reported “starts” for the ovarian versus brain cDNAs. A hybrid luciferase gene directed by rat FGF‐2 5′‐flanking DNA (−1,058/+54) was expressed in rat glioma C6, heart myoblast H9c2, and human astrocytoma U87‐MG cells after gene transfer. The level of transfected FGF‐2 promoter activity was higher in glial cells (C6 and U87‐MG) compared with nonglial (H9c2) cells. Also, expression of this hybrid FGF‐2/luciferase gene was increased in response to phorbol ester or serum treatment of C6 cells. Deletion analysis revealed the presence of both positive and negative regulatory regions that are involved in the transcriptional control of rat FGF‐2 gene by mitogenic stimuli.
Assembly Properties of Amino‐ and Carboxyl‐Terminally Truncated Neurofilament NF‐H Proteins with NF‐L and NF‐M in the Presence and Absence of Vimentin
Authors: Sun Dongming , Macioce Pompeo , Chin Steven S. M. , Liem Ronald K. H.
Published: July 2010

Abstract: To understand the assembly characteristics of the high‐molecular‐weight neurofilament protein (NF‐H), carboxyl‐ and amino‐terminally deleted NF‐H proteins were examined by transiently cotransfecting mutant NF‐H constructs with the other neurofilament triplet proteins, low‐ and middle‐molecular‐weight neurofilament protein (NF‐L and NF‐M, respectively), in the presence or absence of cytoplasmic vimentin. The results confirm that NF‐H can coassemble with vimentin and NF‐L but not with NF‐M into filamentous networks. Deletions from the amino‐terminus show that the N‐terminal head is necessary for the coassembly of NF‐H with vimentin, NF‐L, or NF‐M/vimentin. However, headless NF‐H or NF‐H from which the head and a part of the rod is removed can still incorporate into an NF‐L/vimentin network. Deletion of the carboxyl‐terminal tail of NF‐H shows that this region is not essential for coassembly with vimentin but is important for coassembly with NF‐L into an extensive filamentous network. Carboxyl‐terminal deletion into the α‐helical rod results in a dominant‐negative mutant, which disrupts all the intermediate filament networks. These results indicate that NF‐L is the preferred partner of NF‐H over vimentin and NF‐M, the head region of NF‐H is important for the formation of NF‐L/NF‐H filaments, and the tail region of NF‐H is important to form an extensive network of NF‐L/NF‐H filaments.