bluuebunny/crossref_metadata_2025 huggingface dataset:
Increasing Age Alters Transbilayer Fluidity and Cholesterol Asymmetry in Synaptic Plasma Membranes of Mice
Authors: Igbavboa Urule , Avdulov Nicolai A. , Schroeder Friedhelm , Wood W. Gibson
Published: July 2010

Abstract: Previous studies examining age differences in membrane fluidity and cholesterol content have reported on the average or total change in membrane structure, respectively. However, a membrane consists of an exofacial leaflet and a cytofacial leaflet that differ in fluidity and cholesterol distribution. The purpose of the present experiments was to determine fluidity and cholesterol distribution of the exofacial and cytofacial leaflets of brain synaptic plasma membranes (SPMs) from 3–4‐, 14–15‐, and 24–25‐month‐old C57BL/6NNIA mice by using trinitrobenzenesulfonic acid (TNBS)‐quenching techniques and fluorescent probes. The exofacial leaflet of SPMs from young mice was significantly more fluid compared with the cytofacial leaflet. The large difference in fluidity between the two leaflets was abolished in SPMs of the oldest age group. Total SPM cholesterol and the cholesterol‐to‐phospholipid molar ratio did not differ among the three different age groups of mice. However, considerable differences were observed in the distribution of cholesterol in the two SPM leaflets. The exofacial leaflet contained substantially less cholesterol than did the cytofacial leaflet (13 vs. 87%, respectively) in SPMs of young mice. This asymmetric distribution of cholesterol was significantly modified with increasing age. There was an approximately twofold increase in exofacial leaflet cholesterol in the oldest group compared with the youngest age group. Transbilayer fluidity and cholesterol asymmetry were altered in SPMs of older mice. This approach is a new and different way of viewing how aging modifies membrane structure. Age differences in SPM leaflet structure may be an important factor regulating activity of certain membrane proteins.
The Differential Response of Protein Kinase A to Cyclic AMP in Discrete Brain Areas Correlates with the Abundance of Regulatory Subunit II
Authors: Ventra Carmelo , Porcellini Antonio , Feliciello Antonio , Gallo Adriana , Paolillo Mayra , Mele Evelina , Avvedimento Vittorio Enrico , Schettini Gennaro
Published: July 2010

Abstract: We analyzed the expression and relative distribution of mRNA for the regulatory subunits (RIα, RIIα, and RIIβ) and of 150‐kDa RIIβ‐anchor proteins for cyclic AMP (cAMP)‐dependent protein kinase (PKA) into discrete brain regions. The subcellular distribution of both holoenzyme and free catalytic subunit was evaluated in the same CNS areas. In the neocortex and corpus striatum high levels of RIIβ paralleled the presence of specific RII‐anchoring proteins, high levels of membrane‐bound PKA holoenzyme, and low levels of cytosolic free catalytic activity (C‐PKA). Conversely, in brain areas showing low RIIβ levels (cerebellum, hypothalamus, and brainstem) we found an absence of RII‐anchoring proteins, low levels of membrane‐bound holoenzyme PKA, and high levels of cytosolic dissociated C‐PKA. Response to cAMP stimuli was specifically evaluated in the neocortex and cerebellum, prototypic areas of the two different patterns of PKA distribution. We found that cerebellar holoenzyme PKA was highly sensitive to cAMP‐induced dissociation, without, however, a consistent translocation of C‐PKA into the nucleus. In contrast, in the neocortex holoenzyme PKA was mainly in the undissociated state and poorly sensitive to cAMP. In nuclei of cortical cells cAMP stimulated the import of C‐PKA and phosphorylation of cAMP‐responsive element binding protein. Taken together, these data suggest that RIIβ (whose distribution is graded throughout the CNS, reaching maximal expression in the neocortex) may represent the molecular cue of the differential nuclear response to cAMP in different brain areas, by controlling cAMP‐induced holoenzyme PKA dissociation and nuclear accumulation of catalytic subunits.
Embryonic Expression of Vasoactive Intestinal Peptide (VIP) and VIP Receptor Genes
Authors: Waschek James A. , Ellison Julie , Bravo Dawn T. , Handley Vance
Published: July 2010

Abstract: Vasoactive intestinal peptide (VIP) exhibits pronounced effects on the growth rate of cultured mouse embryonic day (E) 9.5 embryos and acts in tissue culture as a potent glial mitogen and neuron survival factor. However, previous studies using immunohistochemistry or in situ hybridization in the rat have not revealed the presence and location of VIP or VIP mRNA in the early developing embryo CNS. Using a sensitive in situ hybridization assay with a 33P‐labeled riboprobe, we show here that the VIP gene is expressed at least as early as E11 in the mouse hindbrain. Northern blot analysis on RNA from brain dissected from mouse embryos beginning at E14 confirmed that a correct‐size mRNA for VIP was present by E14 and at later time points. Expression of the VIP2 receptor gene was also detected by northern analysis in E14 mouse brains. These studies support the hypothesis that VIP produced by the embryo exerts important effects on embryonic nervous system development.
Molecular Analysis of the Presenilin 1 (S182) Gene in “Sporadic” Cases of Alzheimer's Disease: Identification and Characterisation of Unusual Splice Variants
Authors: Anwar Rashida , Moynihan Terry P. , Ardley Helen , Brindle Nicholas , Coletta P. Louise , Cairns Nigel , Markham Alexander F. , Robinson Philip A.
Published: July 2010

Abstract: Mutations of the presenilin 1 (PS‐1) gene at the Alzheimer's disease (AD) FAD3 locus on chromosome 14q24.3 are responsible for the majority of familial early‐onset AD. As genes responsible for familial forms of AD are obvious candidates for further investigation in “sporadic” disease, we performed a molecular analysis of PS‐1 transcripts extracted from brain tissues of a series of histologically confirmed cases of “sporadic” AD (n = 10) and also from histologically “normal” (non‐Alzheimer) age‐matched brain controls (n = 5). No sequence changes in the PS‐1 coding sequence were detected after analysis by reverse transcription‐PCR. This suggests that the frequency of mutations in the PS‐1 (S182) coding region in “sporadic” Alzheimer's disease is very low. However, we demonstrated that the PS‐1 gene is highly variably spliced. One splice variant involves the 5′ untranslated region of the PS‐1 gene only and hence encodes for normal PS‐1. Six further splice variants involve coding regions of the PS‐1 gene and result in truncated proteins lacking specific transmembrane domains. Most of these variants do not coincide with recognized sites of introns in the PS‐1 gene. One of these variants, resulting in the loss of transmembrane domain TM‐VII, was found only in an AD patient.
Ca2+/Calmodulin‐Dependent Transcriptional Activation of Neuropeptide Y Gene Induced by Membrane Depolarization: Determination of Ca2+‐ and Cyclic AMP/Phorbol 12‐Myristate 13‐Acetate‐Responsive Elements
Authors: Higuchi Hiroshi , Nakano Koichi , Kim Cheol‐Hee , Li Bing‐Sheng , Kuo Che‐Hui , Taira Eiichi , Miki Naomasa
Published: July 2010

Abstract: Membrane depolarization stimuli (high potassium concentration and veratridine) increased neuropeptide Y (NPY) mRNA abundance time‐dependently, without a change in β‐actin mRNA level, in NG108‐15 and PC12 cells. Although the induction by veratridine was blocked completely by tetrodotoxin, the induction by potassium was suppressed minimally. Voltage‐dependent Ca channel blockers and calmodulin antagonists inhibited the increases by both depolarization stimuli completely, suggesting involvement of Ca2+/calmodulin‐dependent kinases (CaM kinases). Transient assay using chloramphenicol acetyltransferase reporter genes containing the rat NPY gene promoter indicated that membrane depolarization and Ca entry stimulate transcription of the NPY gene. The depolarization‐induced transactivation was also blocked by CaM kinase inhibitors. The 200‐bp 5′‐upstream region (−344/−145) was localized as a Ca2+/calmodulin‐responsive element (CaMRE), which confers depolarization‐induced transactivation. It is interesting that this CaMRE did not contain the canonical Ca‐responsive elements such as CRE, SRE, NF‐AT, or the C/EBPβ‐binding site and was separated from a 64‐bp cyclic AMP/phorbol 12‐myristate 13‐acetate‐responsive element (−144/−81). These findings suggested that membrane depolarization regulates the NPY gene transcription positively through the unique CaMRE by activation of CaM kinases following Ca entry through L‐type Ca channels.
Conservation of γ‐Aminobutyric Acid Type A Receptor α6 Subunit Gene Expression in Cerebellar Granule Cells
Authors: Bahn Sabine , Harvey Robert J. , Darlison Mark G. , Wisden William
Published: July 2010

Abstract: The γ‐aminobutyric acid type A receptor cDNAs encoding the α6 subunit homologues from chicken and goldfish have been cloned and sequenced. These proteins exhibit 83 and 75% identity, respectively, to the rat α6 polypeptide. In situ hybridization has demonstrated that, as in mammals, the avian and teleost fish α6 subunit genes are predominately expressed in cerebellar granule cells. Correspondingly, flunitrazepam‐nondisplaceable binding of [3H]Ro 15‐4513 (a benzodiazepine partial inverse agonist), which is a major characteristic of γ‐aminobutyric acid type A receptors that contain the α6 polypeptide, is also mainly found for cerebellar granule cells of fish and chick. The conservation of this expression pattern suggests that γ‐aminobutyric acid type A receptors possessing the α6 subunit are of fundamental importance for cerebellar function and that the corresponding gene regulatory elements, e.g., granule cell‐specific enhancers, have also been conserved.
Estrogens Attenuate and Corticosterone Exacerbates Excitotoxicity, Oxidative Injury, and Amyloid β‐Peptide Toxicity in Hippocampal Neurons
Authors: Goodman Yadong , Bruce Annadora J. , Cheng Bin , Mattson Mark P.
Published: July 2010

Abstract: Steroid hormones, particularly estrogens and glucocorticoids, may play roles in the pathogenesis of neurodegenerative disorders, but their mechanisms of action are not known. We report that estrogens protect cultured hippocampal neurons against glutamate toxicity, glucose deprivation, FeSO4 toxicity, and amyloid β‐peptide (Aβ) toxicity. The toxicity of each insult was significantly attenuated in cultures pretreated for 2 h with 100 nM‐10 µM 17β‐estradiol, estriol, or progesterone. In contrast, corticosterone exacerbated neuronal injury induced by glutamate, FeSO4, and Aβ. Several other steroids, including testosterone, aldosterone, and vitamin D, had no effect on neuronal vulnerability to the different insults. The protective actions of estrogens and progesterone were not blocked by actinomycin D or cycloheximide. Lipid peroxidation induced by FeSO4 and Aβ was significantly attenuated in neurons and isolated membranes pretreated with estrogens and progesterone, suggesting that these steroids possess antioxidant activities. Estrogens and progesterone also attenuated Aβ‐ and glutamate‐induced elevation of intracellular free Ca2+ concentrations. We conclude that estrogens, progesterone, and corticosterone can directly affect neuronal vulnerability to excitotoxic, metabolic, and oxidative insults, suggesting roles for these steroids in several different neurodegenerative disorders.
Evidence for a Role of Calmodulin in Calcium‐Induced Noradrenaline Release from Permeated Synaptosomes: Effects of Calmodulin Antibodies and Antagonists
Authors: Hens Jacques J. H. , Oestreicher A. Beate , De Wit Marina , Marquart Arnoud , Gispen Willem‐Hendrik , De Graan Pierre N. E.
Published: July 2010

Abstract: The nervous tissue‐specific protein B‐50 (GAP‐43), which has been implicated in the regulation of neurotransmitter release, is a member of a family of atypical calmodulin‐binding proteins. To investigate to what extent calmodulin and the interaction between B‐50 and calmodulin are involved in the mechanism of Ca2+‐induced noradrenaline release, we introduced polyclonal anti‐calmodulin antibodies, calmodulin, and the calmodulin antagonists trifluoperazine, W‐7, calmidazolium, and polymyxin B into streptolysin‐O‐permeated synaptosomes prepared from rat cerebral cortex. Anti‐calmodulin antibodies, which inhibited Ca2+/calmodulin‐dependent protein kinase II autophosphorylation and calcineurin phosphatase activity, decreased Ca2+‐induced noradrenaline release from permeated synaptosomes. Exogenous calmodulin failed to modulate release, indicating that if calmodulin is required for vesicle fusion it is still present in sufficient amounts in permeated synaptosomes. Although trifluoperazine, W‐7, and calmidazolium inhibited Ca2+‐induced release, they also strongly increased basal release. Polymyxin B potently inhibited Ca2+‐induced noradrenaline release without affecting basal release. It is interesting that polymyxin B was also the only antagonist affecting the interaction between B‐50 and calmodulin, thus lending further support to the hypothesis that B‐50 serves as a local Ca2+‐sensitive calmodulin store underneath the plasma membrane in the mechanism of neurotransmitter release. We conclude that calmodulin plays an important role in vesicular noradrenaline release, probably by activating Ca2+/calmodulin‐dependent enzymes involved in the regulation of one or more steps in the release mechanism.
Blockade of FG 7142‐Induced Increased Dopamine Utilization by the Glycine/NMDA Receptor Antagonist (+)‐HA 966
Authors: Horger Brian A. , Elsworth John D. , Roth Robert H.
Published: July 2010

Abstract: Systemic administration of the anxiogenic benzodiazepine inverse agonist FG 7142 has been shown to increase selectively dopamine utilization in the medial prefrontal cortex and the shell, but not core, subregion of the nucleus accumbens. In the present study, we examined the functional interaction between benzodiazepine and N‐methyl‐d‐aspartate receptor influences on dopamine utilization in these areas. Male Sprague‐Dawley rats were pretreated with the glycine receptor antagonist (+)‐HA 966 (15 mg/kg, i.p.) or saline 15 min before FG 7142 (20 mg/kg, i.p.) or vehicle administration. Subjects were killed 30 min later and assayed for tissue concentrations of dopamine and its major metabolite 3,4‐dihydroxyphenylacetic acid in the core and shell subdivisions of the nucleus accumbens and the medial prefrontal cortex. (+)‐HA 966 administration blocked FG 7142‐induced increased dopamine utilization in both the medial prefrontal cortex and the shell subdivision of the nucleus accumbens. Results are discussed in terms of N‐methyl‐d‐aspartate receptor influences on the response of mesoaccumbal dopamine neurons to stress.
Intrastriatal Infusion of (±)‐S‐Nitroso‐N‐Acetylpenicillamine Releases Vesicular Dopamine via an Ionotropic Glutamate Receptor‐Mediated Mechanism: An In Vivo Microdialysis Study in Chloral Hydrate‐Anesthetized Rats
Authors: West Anthony R. , Galloway Matthew P.
Published: July 2010

Abstract: The existence of both nitric oxide synthase (NOS) immunoreactive interneurons and amino acid neurotransmitter‐mediated nitric oxide (NO) release in the striatum suggests a role for NO in modulating striatal function. To explore the potential interaction between NO and dopaminergic neurotransmission, the NO‐releasing agent (±)‐S‐nitroso‐N‐acetylpenicillamine (SNAP) was administered locally into the anterior medial striatum of chloral hydrate‐anesthetized rats. SNAP, at 0.5, 1, and 2 mM concentrations, elevated striatal extracellular (EC) dopamine (DA) to 200 ± 42, 472 ± 120, and 2,084 ± 496%, respectively, above baseline levels. Perfusion with (±)‐penicillamine (PEN, 1 mM), the non‐NO‐containing carrier component of SNAP, was ineffective, indicating that PEN is not responsible for SNAP‐mediated DA release. Additional microdialysis experiments suggest SNAP‐mediated DA release is not due to NO‐induced neurotoxicity or blockade of the DA transporter. The DA‐releasing effect of SNAP was attenuated under calcium‐free conditions and abolished in rats pretreated with reserpine (5 mg/kg), implicating a calcium‐sensitive vesicular‐dependent release process. To determine the mechanism of SNAP‐mediated DA release, the guanylyl cyclase (GC) inhibitor LY 83583 (100 µM) was administered 100 min before and during the SNAP pulse. LY 83583 elevated EC DA levels approximately fivefold and potentiated the DA‐releasing effect of SNAP to 2,598 ± 551% above basal DA levels. Similar pretreatments with both the noncompetitive N‐methyl‐d‐aspartate (NMDA) antagonist MK‐801 (10 µM) and the competitive NMDA‐receptor antagonist (±)‐3‐(carboxypiperazin‐4‐yl)propyl‐1‐phosphonic acid [(±)‐CPP, 100 µM] blocked SNAP‐mediated DA release. SNAP‐mediated DA release was also significantly blunted by pretreatment and coperfusion with MgSO4 (10 mM) and 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX, 10 µM) but not (+)‐2‐amino‐3‐phosphonopropionic acid (AP‐3, 10 µM). These results suggest that NO releases DA via a calcium‐sensitive vesicular‐dependent process that is independent of GC activation. In addition, NMDA and kainate/(±)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor‐mediated mechanisms are implicated in NO‐induced DA release.
Nature of Inhibition of Mitochondrial Respiratory Complex I by 6‐Hydroxydopamine
Authors: Glinka Y. , Tipton K. F. , Youdim M. B. H.
Published: July 2010

Abstract: The catecholaminergic neurotoxin 6‐hydroxydopamine causes parkinsonian symptoms in animals and it has been proposed that reactive oxygen species and oxidative stress, enhanced by iron, may play a key role in its toxicity. The present results demonstrate that 6‐hydroxydopamine reversibly inhibits complex I (NADH dehydrogenase) of brain mitochondrial respiratory chain in isolated mitochondria. 6‐Hydroxydopamine itself, rather than its oxidative products, was responsible for the inhibition. Iron(III) did not enhance inhibition but decreased it by stimulating the nonenzyme oxidation of 6‐hydroxydopamine. Inhibition was potentiated to some extent by calcium ion. Desferrioxamine protected complex I activity against the inhibition, but it was not due to its chelator or antioxidative properties. Desferrioxamine was also shown to activate NADH dehydrogenase in the absence of 6‐hydroxydopamine. Activation of mitochondrial respiration by desferrioxamine may contribute to the enhanced neuron survival in the presence of desferrioxamine in some neurodegenerative conditions.
Prosaposin Facilitates Sciatic Nerve Regeneration In Vivo
Authors: Kotani Yasunori , Matsuda Seiji , Sakanaka Masahiro , Kondoh Keiji , Ueno Shu‐ichi , Sano Akira
Published: July 2010

Abstract: Prosaposin, a multifunctional protein, is the precursor of saposins, which activate sphingolipid hydrolases. In addition to acting as a precursor for saposins, prosaposin has been shown to rescue hippocampal CA1 neurons from lethal ischemic damage in vivo and to promote neurite extension of neuroblastoma cells in vitro. Here we show that prosaposin, when added to a collagen‐filled nerve guide after sciatic nerve transection in guinea pigs, increased dramatically the number of regenerating nerve fibers within the guide. To identify the target neurons of prosaposin during peripheral nerve regeneration, we determined the degree of atrophy and chromatolysis of neurons in the spinal anterior horn and dorsal root ganglia on the prosaposin‐treated and untreated side. The effect of prosaposin on large spinal neurons and small neurons of the dorsal root ganglion was more conspicuous. Subsequent immunohistochemistry demonstrated that the atrophy of cholinergic large neurons in the anterior horn is prevented to significant extent by prosaposin treatment. These findings suggest that prosaposin promotes peripheral nerve regeneration by acting on α‐motor neurons in the anterior horn and on small sensory neurons in the dorsal root ganglion. The present study raises the possibility of using prosaposin as a tool for the treatment of peripheral nerve injuries.
Influence of Host Cell Infiltration on the Glycolipid Content of Mouse Brain Tumors
Authors: Seyfried Thomas N. , El‐Abbadi Mohga , Ecsedy Jeffrey A. , Bai Hong Wei , Yohe Herbert C.
Published: July 2010

Abstract: Previous studies showed that levels of some glycosphingolipids (GSLs) expressed in solid brain tumors grown in vivo were reduced or undetectable in cultured cells prepared from the tumors. This phenomenon has been attributed either to suppressed glycolipid synthesis from unknown forces of the tissue culture environment or to the absence of host cells that normally infiltrate the solid tumors growing in vivo. To test further the host cell hypothesis, we examined host cell markers in two experimental mouse brain tumors, the ependymoblastoma and the CT‐2A, that were grown as subcutaneous solid tumors in the flank of C57BL/6J (B6) mice or as cultured cells in vitro. The markers included ganglioside N‐glycolylneuraminic acid (NeuGc), GA1 (asialo‐GM1), and Fc receptor‐bearing cells. NeuGc‐containing gangliosides, GA1, and Fc receptors are expressed by macrophages and lymphoid‐type cells of the mouse host immune system but are not normally expressed by mouse neural cells. Differences in the relative content of Fc receptor‐bearing cells in ependymoblastoma and CT‐2A tumors grown in vivo (8.3 and 16.8%, respectively) were proportional to differences in the relative content of NeuGc‐containing gangliosides (25.5 and 45.1%) and GA1 (8.5 and 13.8%), respectively. Neither cultured tumor cell line expressed Fc receptors, GA1, or NeuGc‐containing gangliosides. These findings suggest that non‐neoplastic host infiltrating cells (macrophages) contribute significantly to the GSL composition of solid tumors growing in vivo.
Membrane Currents Induced in Xenopus Oocytes by the C‐Terminal Fragment of the β‐Amyloid Precursor Protein
Authors: Fraser S. P. , Suh Y.‐H. , Chong Y. H. , Djamgoz M. B. A.
Published: July 2010

Abstract: There is mounting evidence that at least some of the neurotoxicity associated with Alzheimer's disease (AD) is due to proteolytic fragments of the β‐amyloid precursor protein (βAPP). Most research has focused on the amyloid β protein (Aβ), which has been shown to possess ion channel activity. However, the possible role of other cleaved products of the βAPP is less clear. We have investigated the ability of various products of βAPP to induce membrane ion currents by applying them to Xenopus oocytes, a model system used extensively for investigating electrophysiological aspects of cellular, including neuronal, signalling. We focussed on the 105‐amino‐acid C‐terminal fragment (CT105) (containing the full sequence Aβ), which has previously been found to be toxic to cells, although little is known about its mode of action. We have found that CT105 is exceedingly potent, with a threshold concentration of 100–200 nM, in inducing nonselective ion currents when applied from either outside or inside the oocyte and is more effective than either βAPP or the Aβ fragments, β25–35 or β1–40. The ion channel activity of CT105 was concentration dependent and blocked by a monoclonal antibody to Aβ. These results suggest the possible involvement of CT105 in inducing the neural toxicity characteristic of AD.
Early Events in Free Radical‐Mediated Damage of Isolated Nerve Terminals: Effects of Peroxides on Membrane Potential and Intracellular Na+ and Ca2+ Concentrations
Authors: Tretter László , Adam‐Vizi Vera
Published: July 2010

Abstract: The effects of peroxides were investigated on the membrane potential, intracellular Na+ ([Na+]i) and intracellular Ca2+ ([Ca2+]i) concentrations, and basal glutamate release of synaptosomes. Both H2O2 and the organic cumene hydroperoxide produced a slow and continuous depolarization, parallel to an increase of [Na+]i over an incubation period of 15 min. A steady rise of the [Ca2+]i due to peroxides was also observed that was external Ca2+ dependent and detected only at an inwardly directed Ca2+ gradient of the plasma membrane. These changes did not correlate with lipid peroxidation, which was elicited by cumene hydroperoxide but not by H2O2. Resting release of glutamate remained unchanged during the first 15 min of incubation in the presence of peroxides. These alterations may indicate early dysfunctions in the sequence of events occurring in the nerve terminals in response to oxidative stress.
Novel Endogenous Protein Kinase C Substrate Proteins, Neutrinins, in Brain Synaptic Membranes of Maturing Rat
Authors: Chen Xiu‐fang , Miao Hong‐hua , Zhang Jian‐wei , Han Yifan , Du Yu‐cang
Published: July 2010

Abstract: A new family of membrane phosphoproteins designated as P9, P12, P15, P16, and P20 with corresponding apparent molecular weights of 9K, 12K, 15K, 16K, and 20K was characterized from rat brain by using in vitro exogenous or endogenous phosphorylation and autoradiography. As the phosphorylation was selectively inhibited by the protein kinase C (PKC) inhibitor PKC19–31 or Ca2+‐chelating reagents and again stimulated by the PKC activator phorbol 12,13‐dibutyrate, these proteins are thought to be the natural PKC substrates. Because P12, P15, P16, and P20 were neutral proteins (pl 7.0) and specifically distributed in neuronal membranes, the new family of membrane‐associated PKC substrate proteins was referred to as neutrinins. Neutrinins were widely distributed in rat brain, being especially plentiful in the spinal cord, medulla oblongata, cerebellum, and midbrain, relatively scanty in the cerebral cortex, but lacking in cytosol of brain areas and cell membrane preparations of peripheral tissues. The expression of the developmental changes of neutrinins has been monitored by the in vitro exogenous phosphorylation approach, i.e., adding purified PKC to a deactivated synaptosomal plasma membrane system. Levels of all the neutrinin proteins in rat cerebral cortex, as represented by P12, P15, and P16, showed an ontogenetic increase from the early postnatal days to the adult. This appears to be correlated with the commencement of synaptogenesis.
Induction of Nitric Oxide Synthase Inhibits Gap Junction Permeability in Cultured Rat Astrocytes
Authors: Bolaños Juan P. , Medina José M.
Published: July 2010

Abstract: Nitric oxide (•NO) synthase (NOS) was induced in cultured rat astrocytes by incubation with lipopolysaccharide (LPS) for 18 h and gap junction permeability was assessed by the scrape‐loading/Lucifer yellow transfer technique. Induction of NOS was confirmed by determining either the NG‐methyl‐l‐arginine (NMMA)‐inhibitable production of nitrites and nitrates or the conversion of l‐[3H]arginine to l‐[3H]citrulline. Incubation with LPS dose‐dependently inhibited gap junction permeability to 63.3% at 0.05 µg/ml LPS and no further inhibition was observed on increasing the LPS concentration up to 0.5 µg/ml. LPS‐mediated gap junction inhibition was irreversible but was prevented by incubation with the NOS inhibitor NMMA and with the superoxide anion (O2•−) scavenger superoxide dismutase. Incubation of the cells with both the •NO donor S‐nitroso‐N‐acetylpenicillamine and the O2•−‐generating system xanthine/xanthine oxidase inhibited gap junction permeability. These results suggest that the in situ reaction between •NO and O2•−, to form the peroxynitrite anion (ONOO−), may be responsible for the inhibition of gap junction permeability. Scavenging the ONOO− derivative hydroxyl radical (•OH) with either dimethyl sulfoxide or mannitol prevented the LPS‐mediated inhibition of gap junction permeability. Finally, exposure of astrocytes to authentic ONOO− caused a dose‐dependent inhibition of gap junction permeability (65.7% of inhibition at 0.5 mM ONOO−). The pathophysiological relevance of ONOO−‐mediated inhibition of gap junctional communication in astrocytes after NOS induction by LPS is discussed, stressing the possible role played by this mechanism in some neurodegenerative diseases.
Regulation of the Manganese Superoxide Dismutase and Inducible Nitric Oxide Synthase Gene in Rat Neuronal and Glial Cells
Authors: Kifle Y. , Monnier J. , Chesrown S. E. , Raizada M. K. , Nick H. S.
Published: July 2010

Abstract: Bidirectional communication occurs between neuroendocrine and immune systems through the action of various cytokines. Responses to various inflammatory mediators include increases in intracellular reactive oxygen species (ROS), notably, superoxide anion (O2−) and nitric oxide (NO•). Neurotoxicity mediated by NO• may result from the reaction of NO• with O2, leading to formation of peroxynitrite (ONOO−). ROS are highly toxic, potentially contributing to extensive neuronal damage. We, therefore, evaluated the effects of a variety of inflammatory mediators on the regulation of mRNA levels for manganese superoxide dismutase (MnSOD) and inducible nitric oxide synthase (iNOS) in primary cultures of rat neuronal and glial cells. To determine age‐dependent variation of mRNA expression, we used glial cells derived from newborn, 3‐, 21‐, and 95‐day‐old rat brains. Interleukin‐1β, interferon‐γ (IFN‐γ), bacterial lipopolysaccharide (LPS), and tumor necrosis factor‐α showed significant induction of MnSOD in both glial and neuronal cells. However, only LPS and IFN‐γ increased iNOS mRNA. These data demonstrate that these two genes are similarly regulated in two cells of the nervous system, further suggesting that the oxidative state of a cell may dictate a neurotoxic or neuroprotective outcome.
Neuronal Metabolism of Branched‐Chain Amino Acids: Flux Through the Aminotransferase Pathway in Synaptosomes
Authors: Yudkoff Marc , Daikhin Yevgeny , Nelson David , Nissim Itzhak , Erecińska Maria
Published: July 2010

Abstract: The metabolism of branched‐chain amino acids (BCAAs) was studied in cortical synaptosomes. With [15N]leucine (1 mM) as precursor, the cumulative appearance of 15N in [15N]glutamate and [15N]aspartate was 0.2 nmol/min/mg of protein without supplemental α‐ketoglutarate and 0.3 nmol/min/mg of protein in the presence of α‐ketoglutarate (0.5 mM). The BCAA aminotransferase reaction also proceeded in the “reverse” direction [α‐ketoisocaproate (KIC) + glutamate → leucine + α‐ketoglutarate]. This was documented by incubating synaptosomes with [15N]glutamate and measuring the formation of [15N]leucine. Without KIC in the medium, the rate of [15N]leucine production was 0.13 nmol/min/mg of protein. In the presence of 25 µM KIC the rate was 0.79 nmol/min/mg of protein and even greater (1.0 nmol/min/mg of protein) in the presence of 500 µM KIC. The reamination of KIC was two‐ to threefold faster with [2‐15N]glutamine as precursor compared with [15N]glutamate. The ketoacid of valine, α‐ketoisovalerate (KIV), was reaminated to [15N]valine at a rate comparable to that observed with respect to KIC. The BCAA transaminase mediated not only the bidirectional transfer of amino groups between leucine or valine and glutamate, but also the direct transfer of nitrogen between leucine and valine. This was ascertained in studies in which the incubation medium was supplemented with either [15N]leucine and KIV or [15N]valine and KIC (amino acids at 1 mM and ketoacids at 25 or 500 µM). The rate was faster in the direction of leucine formation at both the lower (6.1‐fold) and higher (1.7‐fold) KIC concentration. It is suggested that in synaptosomes the BCAA transaminase (a) functions predominantly in the direction of leucine formation and (b) maintains a constant ratio of BCAAs and ketoacids to one other.
Effects of Chronic Ethanol Consumption and Aging on Dopamine, Serotonin, and Metabolites
Authors: Woods James M. , Druse Mary J.
Published: July 2010

Abstract: This study examined the hypothesis that chronic ethanol consumption results in significant abnormalities in both the dopaminergic and the serotonergic system of aged rats. Levels of dopamine (DA), serotonin [5‐hydroxytryptamine (5‐HT)], 3,4‐dihydroxyphenylacetic acid (DOPAC), and 5‐hydroxyindole‐3‐acetic acid (5‐HIAA) were determined in brain areas of both the nigrostriatal and mesocorticolimbic DA systems in 5‐, 14‐, and 24‐month‐old male Fischer 344 rats. Aging was associated with a reduced concentration of DA in the striatum (ST), ventral tegmental area (VTA), and ventral pallidum (VP) and an increased concentration of 5‐HIAA in the ST, globus pallidus, nucleus accumbens, frontal cortex, and VP. In addition, there was an increase in the 5‐HIAA/5‐HT ratio in all brain areas analyzed. Six weeks of ethanol consumption was accompanied by significant changes in mesocorticolimbic brain areas. In the VTA of 5‐month‐old ethanol‐fed rats DA content was decreased to the levels found in aged rats, e.g., 24 months of age. Ethanol also significantly lowered 5‐HT and 5‐HIAA contents in the VTA and reduced DOPAC and 5‐HIAA levels in the VP. In addition, ethanol blunted the normal age‐related increase in 5‐HIAA/5‐HT ratio in the VTA, VP, and substantia nigra. It is interesting that although the age‐related changes were found in both nigrostriatal and mesocorticolimbic brain areas, the ethanol‐associated effects were found only in brain areas of the mesocorticolimbic system. The changes in DA and 5‐HT function that accompany aging and ethanol consumption may contribute to the problems in motor function and ethanol abuse found in the aged.
A Hydrophilic Peptide Comprising 18 Amino Acid Residues of the Prosaposin Sequence Has Neurotrophic Activity In Vitro and In Vivo
Authors: Kotani Yasunori , Matsuda Seiji , Wen Tong‐Chun , Sakanaka Masahiro , Tanaka Junya , Maeda Nobuji , Kondoh Keiji , Ueno Shu‐ichi , Sano Akira
Published: July 2010

Abstract: Prosaposin, a 517‐amino‐acid glycoprotein, not only acts as the precursor of saposin A, B, C, and D but also possesses neurotrophic activity to rescue hippocampal CA1 neurons from ischemic damage in vivo and to promote neurite extension of neuroblastoma cells in vitro. Recently, the trophic activity of prosaposin on human neuroblastoma cells has been shown to reside in the NH2‐terminal hydrophilic sequence (LIDNNRTEEILY) of the human saposin C. Here we show that prosaposin, saposin C, and a peptide comprising the 18‐amino‐acid sequence (18‐mer peptide; LSELIINNATEELLIKGL) located in the NH2‐terminal hydrophilic sequence of the rat saposin C‐domain promoted survival and neurite outgrowth of cultured rat hippocampal neurons in a dose‐dependent manner. Moreover, infusion for 7 days of the 18‐mer peptide into the lateral ventricle of gerbils, starting either 2 h before or immediately after 3 min of forebrain ischemia, protected ischemia‐induced learning disability and hippocampal CA1 neuronal loss. Thus, we ascribe the in vitro and in vivo trophic actions of prosaposin on hippocampal neurons to the linear 18‐mer sequence and raise the possibility that this peptide can be used as an agent for the treatment of forebrain ischemic damage.
Generation and Characterisation of Stable Cell Lines Expressing Recombinant Human N‐Methyl‐d‐Aspartate Receptor Subtypes
Authors: Grimwood Sarah , Le Bourdellès Béatrice , Atack John R. , Barton Cheryl , Cockett Wendy , Cook Susan M. , Gilbert Elizabeth , Hutson Peter H. , McKernan Ruth M. , Myers Jan , Ragan C. Ian , Wingrove Peter B. , Whiting Paul J.
Published: July 2010

Abstract: Transfection of mouse L(tk‐) cells with human N‐methyl‐d‐aspartate (NMDA) receptor subunit cDNAs under the control of a dexamethasone‐inducible promoter has been used to generate two stable cell lines expressing NR1a/NR2A receptors and a stable cell line expressing NR1a/NR2B receptors. The cell lines have been characterised by northern and western blot analyses, and the pharmacology of the recombinant receptors determined by radioligand binding techniques. Pharmacological differences were identified between the two NMDA receptor subtypes. The glutamate site antagonist d,l‐(ε)‐2‐[3H]amino‐4‐propyl‐5‐phosphono‐3‐pentanoic acid ([3H]CGP 39653) had high affinity for NR1a/NR2A receptors (KD = 3.93 nM) but did not bind to NR1a/NR2B receptors. Glycine site agonists showed a 2.6–5.4‐fold higher affinity for NR1a/NR2B receptors. Data from radioligand binding studies indicated that one of the cell lines, NR1a/NR2A‐I, expressed a stoichiometric excess of the NR1a subunit, which may exist as homomeric assemblies. This observation has implications when interpreting data from pharmacological analysis of recombinant receptors, as well as understanding the assembly and control of expression of native NMDA receptors.
Promoter Region and Alternatively Spliced Exons of the Rat μ‐Opioid Receptor Gene
Authors: Mayer P. , Schulzeck S. , Kraus J. , Zimprich A. , Höllt V.
Published: July 2010

Abstract: The actions of exogenous and endogenous opioids are mediated by at least three different opioid receptors, called μ, κ, and δ. Recently, we have detected a new variant of the rat μ‐opioid receptor, which we termed rMOR1B and which differs from rMOR1 (now also called rMOR1A) in the amino acid sequence at the C‐terminus. Both isoforms were proposed to be splicing variants of the same gene. To elucidate the molecular mechanism leading to the formation of the new variant, the exon/intron structure of the rat μ‐opioid receptor gene in the respective area has been determined by analyzing a genomic P1 phage clone. In addition, we have investigated the putative promoter region of this gene. The present study revealed that rMOR1B is generated by an alternative splicing event whereby a previously unknown exon will be placed behind exon 3 to form rMOR1B mRNA, which is separated from the latter by an intron. Therefore, this new exon has to be called exon 4, whereas the former exon 4, which encodes the C‐terminus of MOR1A, now becomes exon 5. Examination of the putative rat promoter region revealed a high degree of nucleotide sequence homology to the mouse gene. Using an RNase protection approach, one single transcription initiation site could be located at 230 bp upstream of the translation start. This is similar to the situation in the mouse, where four major transcription start sites were reported to lie close together around 270 bp upstream of the protein coding region.
Induction of the Serotonin1A Receptor in Neuronal Cells During Prolonged Stress and Degeneration
Authors: Singh Jyoti K. , Chromy Brett A. , Boyers Michael J. , Dawson Glyn , Banerjee Probal
Published: July 2010

Abstract: Neuronal migration in brain is followed by differentiation of committed neurons and simultaneous apoptosis of uncommitted preneuronal cells due to a limiting supply of trophic factors and nutrients. We have dissected differentiation and apoptosis by designing a simple in vitro model for this nutrient deprivation using engineered neuronal cell lines stably transfected with a promoterless segment (G‐21) of the intronless human serotonin1A receptor (5‐HT1A‐R) gene. Despite the use of widely different heterologous promoters (cytomegalovirus and Rous sarcoma virus) for the stable expression of G‐21, a dramatic increase in expression of the 5‐HT1A‐R (five‐ to 15‐fold) and its mRNA was always observed during degeneration and apoptosis of nutrient‐deprived neuronal cells. Involvement in this induction of a 170‐bp 5′‐end untranslated sequence (5′‐UT) (tail end of the 500‐bp natural promoter) of G‐21 was confirmed by stable transfection of neuronal cells with an SV‐40 promoter‐driven construct harboring the 5′‐UT and the reporter chloramphenicol acetyltransferase (CAT) cDNA. Presence of the 5′‐UT resulted in a threefold increase in CAT expression during nutrient deprivation in randomly chosen clones. The induction was also observed in the endogenous 5‐HT1A‐R, expressed by embryonic day 16 mouse hippocampal neurons, subsequent to nutrient deprivation and onset of degeneration. A trophic role of the 5‐HT1A‐R has been suggested in earlier studies. Considering the example of protective heat shock proteins, which are induced during various types of stress, our results suggest that stressed neuronal cells undergoing degeneration and apoptosis synthesize increased levels of 5‐HT1A‐R as a final attempt to survive.
Interaction of NMDA and Dopamine D2L Receptors in Human Neuroblastoma SH‐SY5Y Cells
Authors: Nair Venugopalan D. , Niznik Hyman B. , Mishra Ram K.
Published: July 2010

Abstract: To understand the mechanism of interaction of the dopamine D2L receptors with NMDA receptors, we have developed a model by transfecting human neuroblastoma SH‐SY5Y cells with the human dopamine D2L receptor gene. In vitro blockade of NMDA receptors by the specific antagonists MK‐801 and (±)‐3‐(2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonic acid (CPP) on human neuroblastoma SH‐SY5Y cells expressing human dopamine D2L receptors resulted in a significant increase in the density of D2L receptors without a significant change in receptor affinity. Moreover, the dopamine receptor mRNA level increased by ∼50% by the blockade of NMDA with MK‐801. These results suggest a possible interaction of NMDA and dopamine D2L receptors in neuroblastoma SH‐SY5Y cells. This system would serve as an excellent model to study the molecular mechanisms involved in the interaction of these two receptors.
The Phosphoinositide Signal Transduction System Is Impaired in Bipolar Affective Disorder Brain
Authors: Jope Richard S. , Song Ling , Li Peter P. , Young L. Trevor , Kish Stephen J. , Pacheco Mary A. , Warsh Jerry J.
Published: July 2010

Abstract: The function of the phosphoinositide second messenger system was assessed in occipital, temporal, and frontal cortex obtained postmortem from subjects with bipolar affective disorder and matched controls by measuring the hydrolysis of [3H]phosphatidylinositol ([3H]PI) incubated with membrane preparations and several different stimulatory agents. Phospholipase C activity, measured in the presence of 0.1 mM Ca2+ to stimulate the enzyme, was not different in bipolar and control samples. G proteins coupled to phospholipase C were concentration‐dependently activated by guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) and by NaF. GTPγS‐stimulated [3H]PI hydrolysis was markedly lower (50%) at all tested concentrations (0.3–10 µM GTPγS) in occipital cortical membranes from bipolar compared with control subjects. Responses to GTPγS in temporal and frontal cortical membranes were similar in bipolars and controls, as were responses to NaF in all three regions. Brain lithium concentrations correlated directly with GTPγS‐stimulated [3H]PI hydrolysis in bipolar occipital, but not temporal or frontal, cortex. Carbachol, histamine, trans‐1‐aminocyclopentyl‐1,3‐dicarboxylic acid, serotonin, and ATP each activated [3H]PI hydrolysis above that obtained with GTPγS alone, and these responses were similar in bipolars and controls except for deficits in the responses to carbachol and serotonin in the occipital cortex, which were equivalent to the deficit detected with GTPγS alone. Thus, among the three cortical regions examined there was a selective impairment in G protein‐stimulated [3H]PI hydrolysis in occipital cortical membranes from bipolar compared with control subjects. These results directly demonstrate decreased activity of the phosphoinositide signal transduction system in specific brain regions in bipolar affective disorder.
Apolipoprotein E and Low‐Density Lipoprotein Binding and Internalization in Primary Cultures of Rat Astrocytes: Isoform‐Specific Alterations
Authors: Guillaume Daniel , Bertrand Philippe , Dea Doris , Davignon Jean , Poirier Judes
Published: July 2010

Abstract: Apolipoprotein (apo) E is likely involved in redistributing cholesterol and phospholipids during compensatory synaptogenesis in the injured CNS. Three common isoforms of apoE exist in human (E2, E3, and E4). The apoE4 allele frequency is markedly increased in both late‐onset sporadic and familial Alzheimer's disease (AD). ApoE concentration in the brain of AD subjects follows a gradient: ApoE levels decrease as a function of E2 > E3 ≫ E4. It has been proposed that the poor reinnervation capacity reported in AD may be caused by impairment of the apoE/low‐density lipoprotein (LDL) receptor activity. To understand further the role of this particular axis in lipid homeostasis in the CNS, we have characterized binding, internalization, and degradation of human 125I‐LDL to primary cultures of rat astrocytes. Specific binding was saturable, with a KD of 1.8 nM and a Bmax of 0.14 pmol/mg of proteins. Excess unlabeled human LDL or very LDL (VLDL) displaced 70% of total binding. Studies at 37°C confirmed that astrocytes bind, internalize, and degrade 125I‐LDL by a specific, saturable mechanism. Reconstituted apoE (E2, E3, and E4)‐liposomes were labeled with 125I and incubated with primary cultures of rat astrocytes and hippocampal neurons to examine specific binding. Human LDL and VLDL displaced binding and internalization of all apoE isoforms similarly in both astrocytes and neurons. 125I‐ApoE2 binding was significantly lower than that of the other 125I‐apoE isoforms in both cell types. 125I‐ApoE4 binding was similar to that of 125I‐apoE3 in both astrocytes and neurons. On the other hand, 125I‐apoE3 binding was significantly higher in neurons than in astrocytes. These isoform‐specific alterations in apoE‐lipoprotein pathway could explain some of the differences reported in the pathophysiology of AD subjects carrying different apoE alleles.
Thrombin Causes Cell Spreading and Redistribution of β‐Amyloid Immunoreactivity in Cultured Hippocampal Neurons
Authors: Brewer Gregory J.
Published: July 2010

Abstract: Culture of rat embryonic hippocampal neurons in serum‐free B27/Neurobasal for 4 days enabled tests of the effect of added thrombin on differentiated cell morphology and processing of the amyloid precursor protein (APP). By fluorescence microscopy of neurons labeled with dil and by scanning electron microscopy, an increase in spreading of the neuron soma was clearly seen in cells treated with 1 µg/ml (27 nM) of thrombin for 24 h. This treatment also caused a dose‐dependent increase in immunoreactive area/cell, detected with antibody 4G8 binding to the β‐amyloid region of APP. Thrombin treatment also produced a dose‐dependent increase in immunoreactive brightness detected with the Alz‐50 antibody. Thrombin did not affect viability or cause neurite retraction. The thrombin effect on 4G8 immunoreactivity required 24 h for full effect and could be blocked by the thrombin inhibitor antithrombin III or hirudin. A thrombin receptor appeared to be activated because a full immunoreactive response was observed by treatment of neurons with the thrombin receptor‐activating peptide SFLLRNPNNKYEPF. When cytoplasmic extracts were analyzed by western immunoblots or by pulse‐chase radiolabeling, no thrombin‐dependent changes in processing of 127‐ and 120‐kDa bands were seen. Material migrating in the region of synthetic βA4 was not found. Together, these results suggest that thrombin acts on neurons through a thrombin receptor to stimulate cell spreading and redistribution of APP without amyloidogenic changes. The adhesion responsible for this spreading could be important in altering synaptic connections in the brain.
Dopaminergic Regulation of Secretory Granule‐Associated Proteins in Rat Intermediate Pituitary
Authors: Oyarce Ana Maria , Hand Tracey A. , Mains Richard E. , Eipper Betty A.
Published: July 2010

Abstract: The biosynthesis of peptides requires the synthesis of the prohormone, several biosynthetic processing enzymes, and other granule constituents. We have investigated the regulated expression of proopiomelanocortin (POMC) and five enzymes essential for the processing of POMC to smaller, bioactive peptides in intermediate pituitary melanotropes. Rats were treated with a dopaminergic agonist (bromocriptine) or antagonist (haloperidol) for periods ranging from 1 h to 5 days, followed by analyses of mRNA levels and protein biosynthetic rates. Multiplex RNase protection assays showed that bromocriptine treatment caused a striking decrease in POMC mRNA levels, and significant decreases in mRNA levels for prohormone convertase 2 (PC2), carboxypeptidase H (CPH), and peptidylglycine α‐amidating monooxygenase (PAM). Smaller increases in mRNA levels were seen after haloperidol stimulation. Protein biosynthetic rates changed more profoundly than mRNA levels at short drug treatment times, indicating a role for translational effects after treatment with bromocriptine and with haloperidol. The homogeneous population of melanotropes in the intermediate lobe of the pituitary allows a quantitative analysis of transcript levels and biosynthetic rates. POMC mRNA levels are 200–1,000‐fold higher than levels of any of the processing enzyme mRNAs, and POMC biosynthetic rates exceed those of PC2, PC1, and PAM by 1,000–10,000‐fold.