bluuebunny/crossref_metadata_2025 huggingface dataset:
Localisation of Microtubule‐Associated Protein 1B Phosphorylation Sites Recognised by Monoclonal Antibody SMI‐31
Authors: Johnstone Mandy , Goold Robert G. , Bei Di , Fischer Itzhak , Gordon‐Weeks Phillip R.
Published: July 2010

Abstract: MAP 1B is a microtubule‐associated phosphoprotein that is expressed early in neurons and plays a role in axon growth. MAP 1B has two types of phosphoisoforms, one of which is developmentally down‐regulated after neuronal maturation and one of which persists into adulthood. Because phosphorylation regulates MAP 1B binding activity, characterisation of the phosphorylation sites and identification of the corresponding kinases/phosphatases are important goals. We have characterised the developmentally down‐regulated phosphorylation sites recognised by monoclonal antibody (mAb) SMI‐31. We purified MAP 1B from neonatal rat brain and mapped the mAb SMI‐31 sites to specific MAP 1B fragments after chemical cleavage. We then developed an in vitro kinase assay by using a high‐speed spin supernatant from neonatal rat brain in the presence of ATP and recombinant proteins encoding selective regions of the MAP 1B molecule. Phosphorylation of the recombinant protein was detected on western blots using mAb SMI‐31. This analysis showed that mAb SMI‐31 recognises two recombinant proteins corresponding to residues 1,109–1,360 and 1,836–2,076 of rat MAP 1B after in vitro phosphorylation. The former phosphorylation site was further defined in the in vitro kinase assay by inhibition with peptides and antibodies from candidate regions of the MAP 1B sequence. This approach identified a region of 20 amino acids, from 1,244 to 1,264, characterised by a high concentration of serines immediately upstream of prolines, indicating that the kinase responsible is a proline‐directed serine kinase.
Elevation of Cyclic AMP Levels in Astrocytes Antagonizes Cytokine‐Induced Adhesion Molecule Expression
Authors: Ballestas Mary E. , Benveniste Etty N.
Published: July 2010

Abstract: We have examined the effect of elevating cyclic AMP levels on cytokine‐mediated enhancement of intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) gene expression by astrocytes. Treatment of astrocytes with the cyclic AMP mimetic dibutyryl‐cyclic AMP, or the agonists norepinephrine, forskolin, prostaglandin E2, and cholera toxin alone had no effect on ICAM‐1 or VCAM‐1 mRNA gene expression. However, elevating cyclic AMP levels within the cells by these agents suppressed interleukin‐1β‐ and tumor necrosis factor‐α‐induced adhesion molecule expression at both the mRNA and protein levels. The phosphodiesterase type IV inhibitor, rolipram, was able to potentiate the inhibitory effect of forskolin on ICAM‐1 and VCAM‐1 gene expression. Inhibition of tumor necrosis factor‐α‐induced VCAM‐1 mRNA levels by forskolin was partially due to enhanced degradation of VCAM‐1 message, whereas the decay rates of tumor necrosis factor‐α‐induced ICAM‐1 message and interleukin‐1β‐induced ICAM‐1/VCAM‐1 message were not affected by forskolin treatment. These results demonstrate that the pathways used by interleukin‐1β and tumor necrosis factor‐α to induce adhesion molecule expression are antagonized by cyclic AMP‐dependent protein kinase‐mediated signaling pathways.
Microdialysis Study of Modification of Hypothalamic Neurotransmitters in Streptozotocin‐Diabetic Rats
Authors: Ohtani Nobuyo , Ohta Mitsuaki , Sugano Tsukasa
Published: July 2010

Abstract: Neurochemical changes in the ventromedial hypothalamus (VMH) after a single intravenous injection of streptozotocin were examined, using in vivo brain microdialysis under free‐moving conditions. Although streptozotocin‐induced diabetes produced significant decreases in extracellular concentrations of noradrenaline (NA), serotonin (5‐HT), and their metabolites in the VMH, the ratios of 3‐methoxy‐4‐hydroxyphenylglycol/NA and 5‐hydroxyindoleacetic acid (5‐HIAA)/5‐HT were increased. Experimental diabetes led to a pronounced increase in extracellular GABA, which correlated strongly with the decrease in dialysate levels of NA, and to a smaller extent with that of 5‐HT. A modification of dopamine (DA) metabolism was induced in the VMH of diabetic rats, whereas there was no change in dialysate DA levels. Daily injections of insulin were able to restore their levels to normal in the areas tested in the microdialysis study. The equal increases in dialysate 5‐HT and 5‐HIAA and the better restoration of the 5‐HIAA/5‐HT ratio after insulin therapy indicate that serotonergic activity may depend on the levels of circulating insulin more than on noradrenergic activity. Circulating NA was reduced in streptozotocin‐diabetic rats, suggesting that the diabetes‐induced reduction in sympathetic activity is accompanied by decreases in NA, or 5‐HT, or both, in the VMH.
Energetic Dysfunction in Quinolinic Acid‐Lesioned Rat Striatum
Authors: Bordelon Yvette M. , Chesselet Marie‐Françoise , Nelson David , Welsh Frank , Erecińska Maria
Published: July 2010

Abstract: Impairment of mitochondrial energy metabolism may contribute to the selective neuronal degeneration observed in Huntington's disease and other neurodegenerative disorders. Intrastriatal injection of the excitotoxin, quinolinic acid, produces a pattern of neuronal death similar to that seen in Huntington's disease. However, little is known about the effects of quinolinic acid on striatal energetics. In the present work, time‐dependent changes in energy metabolism caused by injection of quinolinic acid into rat striatum were examined. Oxygen consumption by free and synaptic mitochondria was quantified and correlated with the concentrations of nucleotides and amino acids at different times after injection. Compared with saline‐treated controls, a decrease in ADP‐stimulated (state 3) to basal (state 4) oxygen consumption (respiratory control ratio) by free mitochondria was apparent in quinolinic acid‐injected striata as early as 6 h after treatment. No significant changes were seen in nucleotide concentrations at this time. By 12 h after injection, the decline in the respiratory control ratio was more pronounced (45%), and reductions in ATP, NAD, aspartate, and glutamate (30–60%) were also observed. These results show that injection of quinolinic acid in vivo produces progressive mitochondrial dysfunction, which may be a common and critical event in the cell death cascade initiated in Huntington's disease and in animal models of this neurodegenerative disorder. The indicators of mitochondrial function examined in this study, therefore, may be useful in evaluating the efficacy of neuroprotective agents.
Identification of Nitric Oxide‐Sensitive and ‐Insensitive Forms of Cytoplasmic Guanylate Cyclase
Authors: Prabhakar S. , Short D. B. , Scholz N. L. , Goy M. F.
Published: July 2010

Abstract: Cytoplasmic, nitric oxide‐activated guanylate cyclases are expressed in many regions of the mammalian brain and are thought to participate in functions as diverse as synaptogenesis and long‐term potentiation. In this study, we have characterized cytoplasmic guanylate cyclases in the nervous system of an invertebrate, the American lobster. Cytoplasmic cyclase specific activity is higher in lobster nerve cord than in any other lobster tissue tested, and considerably higher than in typical rat tissues (cerebellum, lung, and liver). However, nitric oxide donors have minimal effects on lobster nerve cord cyclic GMP production, when applied either to intact tissue or to cytoplasmic extracts. Parallel immunocytochemical studies, using an anti‐cyclic GMP antibody, reveal that only a small subset of lobster neurons responds to nitric oxide with a significant elevation of cyclic GMP levels. HPLC analysis of nerve cord cytoplasm reveals two chromatographically separable cyclases, a minor nitric oxide‐sensitive form whose retention time is identical to that of the conventional mammalian enzyme and a more abundant nitric oxide‐insensitive form that appears to be novel. The physiological function and phylogenetic distribution of this nitric oxide‐insensitive enzyme, and the signaling mechanisms that regulate its activity, are not known.
Regulation of the Genes Encoding Interleukin‐6, Its Receptor, and gp130 in the Rat Brain in Response to the Immune Activator Lipopolysaccharide and the Proinflammatory Cytokine Interleukin‐1β
Authors: Vallières Luc , Rivest Serge
Published: July 2010

Abstract: Interleukin‐6 (IL‐6) is a pleiotropic cytokine believed to play key roles in the neuroimmune interactions. This molecule may act on the nervous system by interacting with its specific receptor subunit (IL‐6R) and the signal transducer gp130. The purposes of the present study were to describe the central distribution of IL‐6, IL‐6R, and gp130 mRNAs under basal conditions and to verify the influence of the immune activator lipopolysaccharide (LPS) and the proinflammatory cytokine interleukin‐1β (IL‐1β) on the expression of IL‐6 and its related genes throughout the rat brain. Rats were killed at multiple times after intraperitoneal injection of the bacterial endotoxin and intravenous administration of the recombinant rat IL‐1β (rrIL‐1β), and their brains were cut into 30‐µm coronal sections from the olfactory bulb to the end of the medulla. Each transcript was localized by in situ hybridization histochemistry using 35S‐labeled rat riboprobes. The results show that IL‐6 mRNA was undetectable in the brain under basal conditions and following the injection of rrIL‐1β. Injection of LPS rapidly stimulated transcription of this gene in the choroid plexus and the sensorial circumventricular organs (CVOs), including the organum vasculosum laminae terminalis (OVLT), subfornical organ, median eminence, and area postrema. Conversely, IL‐6R and gp130 mRNAs were heterogeneously distributed throughout the brain under basal conditions. The injection of LPS stimulated the biosynthesis of IL‐6R in the CVOs, medial preoptic area, bed nucleus stria terminalis, central nucleus of the amygdala, hippocampus, hypothalamic paraventricular nucleus, cerebral cortex, and blood vessels. Increased levels of IL‐6R mRNA were also observed in the microvasculature following rrIL‐1β injection. Finally, gp130 mRNA expression was increased in the OVLT and throughout the endothelium of brain capillaries of LPS‐treated rats but remained unchanged after administration of rrIL‐1β. These results demonstrate that expression of the genes encoding IL‐6, IL‐6R, and gp130 can be up‐regulated in selective regions of the brain in response to the bacterial endotoxin LPS and the proinflammatory cytokine IL‐1β (only for IL‐6R expression). This fine genetic regulation might be of great importance in the neuroimmune interplay and provides the evidence that sensorial CVOs and microvasculature are in a privileged position to mediate the action of IL‐6 of central and/or systemic origin in the brain of immune‐challenged animals.
3‐Methoxytyramine Formation Following Monoamine Oxidase Inhibition Is a Poor Index of Dendritic Dopamine Release in the Substantia Nigra
Authors: Elverfors Anders , Pileblad Erik , Lagerkvist Sören , Bergquist Filip , Jonason Jan , Nissbrandt Hans
Published: July 2010

Abstract: This study was undertaken, using microdialysis, to compare the extracellular concentration of 3‐methoxytyramine and dopamine in dialysate from the striatum and substantia nigra, after pargyline (75 mg/kg), after pargyline plus amphetamine (3 mg/kg), and after pargyline plus reserpine (5 mg/kg) administration. Treatment with pargyline alone increased the extracellular dopamine concentration by 70% in the striatum and by 140% in the substantia nigra and induced in both regions a time‐dependent accumulation of 3‐methoxytyramine. The addition of d‐amphetamine to pargyline increased the extracellular dopamine concentration, compared with pargyline‐treated controls, to the same extent in both the substantia nigra (maximally by 360%) and the striatum (maximally by 400%), but the concomitant increase of 3‐methoxytyramine accumulation in the dialysate was relatively smaller in the substantia nigra compared with the striatum. Reserpine treatment decreased the extracellular dopamine concentration in both regions below the detection level (<10% of basal value). When pargyline was added to reserpine, the striatal extracellular dopamine concentration increased to 50% of pargyline‐treated controls and the striatal 3‐methoxytyramine accumulation was less than in pargyline‐treated controls. However, in the substantia nigra, the addition of pargyline to reserpine resulted in dopamine concentrations as high as after pargyline only and the 3‐methoxytyramine accumulation was not changed compared with pargyline‐treated controls. In summary, our results indicate that dopamine in the substantia nigra is released from reserpine‐sensitive storage sites and that pargyline‐induced 3‐methoxytyramine accumulation is a poor indicator of the local dopamine release. The latter observation may be explained by the fact that the dopamine‐metabolizing enzyme, catechol‐O‐methyltransferase, is located inter alia in the dopamine‐containing cell bodies/dendrites in the substantia nigra, in contrast to the situation in the terminals in the striatum where catechol‐O‐methyltransferase is located only in nondopaminergic cells.
Characterization of Protein Kinase C in Photoreceptor Outer Segments
Authors: Williams David S. , Liu Xinran , Schlamp Cassandra L. , Ondek Brian , Jaken Susan , Newton Alexandra C.
Published: July 2010

Abstract: Protein kinase C (PKC) has been implicated in regulating several proteins involved in phototransduction. This contribution characterizes the biochemical and immunological properties of PKC isozyme(s) in the photoreceptor outer segment. Activity measurements revealed that at least 85% of the PKC in this specialized compartment belongs to the subfamily of Ca2+‐regulated (conventional) PKCs. Of the known Ca2+‐dependent PKCs, only PKCα was immunodetected by western blot analysis of rod outer segment proteins. However, the ratio of immunoreactivity to enzyme activity for rod outer segment PKC was no more than 40% of that for brain PKC, using antibodies against conventional PKCs. Therefore, at least half the Ca2+/lipid‐stimulated activity in rod outer segment preparations cannot be accounted for by the known isozymes, suggesting the presence of a previously uncharacterized isozyme. Despite extensive tests using a variety of antibodies against different domains of PKCα, PKCα could not be detected in rod outer segments by immunofluorescence of retinal sections. In summary, our data reveal that most of the PKC in photoreceptor outer segments is of the conventional type and that most, if not all, of this conventional PKC activity comes from a novel isozyme(s).
Calcium Mobilization by Ryanodine Promotes the Phosphorylation of Initiation Factor 2α Subunit and Inhibits Protein Synthesis in Cultured Neurons
Authors: Alcázar Alberto , De La Vega Cristina Martín , Bazán Eulalia , Fando Juan L. , Salinas Matilde
Published: July 2010

Abstract: Protein synthesis plays an important role in the viability and function of the cell. There is evidence indicating that Ca2+ may be a physiological regulator of the translational process. In the present study, the effect of agents that increase intracellular calcium levels by different mechanisms, as well as repercussion on the rate of protein synthesis, including phosphorylation of initiation factor 2α subunit, and double‐stranded RNA‐dependent eIF‐2α kinase (PKR) activity were analyzed. Glutamate (100 µM) and K+ (60 mM), which increase intracellular calcium levels (the former mostly by the influx of extracellular calcium via voltage‐sensitive calcium channels, and the latter by receptor‐operated calcium channels), and carbachol (1 mM), as well as glutamate, which mobilizes intracellular calcium from the endoplasmic reticulum via activation of inositol 1,4,5‐trisphosphate receptor, did not modify any of the analyzed parameters. Nevertheless, 100 nM ryanodine, which increases intracellular calcium concentration by activating the ryanodine receptor, promoted a significant decrease in the rate of protein synthesis and increased both initiation factor 2α subunit phosphorylation and PKR activity. From our results, we can conclude that inhibition of protein synthesis is dependent on the mobilization of intracellular calcium from internal stores. Moreover, they strongly suggest that this inhibition is only promoted when calcium is increased via ryanodine receptor, and possibly by activation of PKR activity.
Characterization of Myelin Basic Protein Charge Microheterogeneity in Developing Mouse Brain and in the Transgenic Shiverer Mutant
Authors: Palma Anthony E. , Owh Phillip , Fredric Christopher , Readhead Carol , Moscarello Mario A.
Published: July 2010

Abstract: Myelin basic protein (MBP) is a highly heterogeneous family of membrane proteins consisting of several isoforms resulting from alternative splicing and charge isomers arising from posttranslational modifications. Although well characterized in the bovine and human species, those in the mouse are not. With the availability of a number of transgenic and knockout mice, the need to understand the chemical nature of the MBPs has become very important. To isolate and characterize the MBP species in murine brain, two methods were adapted for use with the small amounts of MBP available from mice. The first was a scaled‐down version of the preparative CM‐52 chromatographic system commonly used to isolate MBP charge isomers; the second was an alkaline‐urea slab gel technique that required five times less material than the conventional tube gel system and, from these gels, western blots were readily obtained. Murine MBP was resolved into two populations of charge isomers: the 18.5‐ and 14‐kDa isoforms. Isolation and characterization of these charge isomers or components permitted us to assign possible posttranslational modifications to some of them. Component 1 (C‐1), the most cationic isomer, had a molecular weight of 14,140.38 ± 0.79. C‐2 consisted of two 14‐kDa species, 14,136.37 ± 0.74 and 14,204.45 ± 0.70. Two variants, 14,215.57 ± 0.94 and 18,413.57 ± 0.76, constituted C‐3. C‐4, C‐5, and C‐8 (the least cationic isomer) each consisted of both 14‐ and 18.5‐kDa isoforms. During myelinogenesis, the 18.5‐kDa isoform appeared first (day 4); the 14‐kDa isoform appeared at day 16 and subsequently became the dominant isoform. The transgenic shiverer mutant synthesized mainly the 18.5‐kDa isoform, but none of the 14‐kDa isoform, similar to the 4‐day‐old mouse. We concluded that the trangenic shiverer was able to initiate myelinogenesis with the 18.5‐kDa isoform, but was unable to complete myelinogenesis because of the absence of the 14‐kDa isoform.
Identification of Kainate Receptor‐Mediated Intracellular Calcium Increases in Cultured Rat Cerebellar Granule Cells
Authors: Savidge Jonathan R. , Bleakman David , Bristow David R.
Published: July 2010

Abstract: The functional expression of the kainate subtype of glutamate receptor (GluR) has been investigated in cultured rat cerebellar granule cells using single cell intracellular calcium ([Ca2+]i) measurements. Both AMPA‐ and kainate‐induced [Ca2+]i increases could be blocked completely by the AMPA receptor‐selective antagonist LY300168 (50 µM). However, following treatment with concanavalin A, an inhibitor of kainate receptor desensitisation, 30% of cells showed a kainate‐induced [Ca2+]i rise of >100 nM in the presence of LY300168. Responses to 30 µM kainate in the presence of LY300168 were virtually abolished by the AMPA and GluR5 kainate receptor competitive antagonist LY293558 (100 µM). These results demonstrate the presence of functional kainate receptors on cultured cerebellar granule cells, and suggest that the GluR5 subtype of kainate receptor plays a significant role in kainate receptor‐mediated [Ca2+]i increases.
Antidiabetic Sulphonylureas Stimulate Acetylcholine Release from Striatal Cholinergic Interneurones Through Inhibition of KATP Channel Activity
Authors: Lee Kevin , Brownhill Varuni , Richardson Peter J.
Published: July 2010

Abstract: The sulphonylureas tolbutamide and glibenclamide were shown to stimulate acetylcholine release from rat striatal slices. To determine the mechanism of this effect, whole‐cell patch‐clamp recordings were made from large neurones within the striatum that displayed morphological, electrophysiological, and pharmacological characteristics typical of cholinergic interneurones. Dialysis of these neurones with a pipette solution containing low concentrations of ATP produced a gradual hyperpolarisation that could be reversed by bath application of the sulphonylureas. In voltage‐clamp studies, these compounds were shown to act through the inhibition of a potassium conductance. It is concluded that cholinergic interneurones within the rat striatum express sulphonylurea‐sensitive ATP‐sensitive potassium channel activity. These channels are probably cytoprotective and may prove to be novel sites of therapeutic modulation.
Chromosomal Localization of the Myelin‐Associated Oligodendrocytic Basic Protein and Expression in the Genetically Linked Neurological Mouse Mutants Ducky and Tippy
Authors: Holz Andreas , Frank Marcus , Copeland Neal G. , Gilbert Debra J. , Jenkins Nancy A. , Schwab Martin E.
Published: July 2010

Abstract: The alternatively spliced cDNAs encoding the myelin‐associated/oligodendrocytic basic proteins (MOBPs) have recently been identified in rat. The Mobp gene maps to the distal part of mouse chromosome 9 at a region syntenic with the human chromosome 3p22‐p21.3. Two nonallelic mouse mutants, tippy and ducky, with severe neurological phenotypes map to the vicinity of the Mobp locus. We therefore tested whether MOBP malfunction could explain the tippy and ducky defects. In tippy mutant animals, MOBP expression and that of other myelin markers were indistinguishable from wild type. The ultrastructure of tippy myelin was shown to be normal. Ducky animals showed a slight reduction of the brain size, most evident in the spinal cord, but normal progress of myelination. Both MOBP and myelin basic protein expression were lowered only regionally in the CNS, but were mostly normal in the anterior parts of the brain. Ultrastructurally, ducky myelin appeared normal. MOBP transcript sizes and the molecular weights of the encoded proteins were shown to be normal in both mutants. Finally, the nucleotide sequence of the abundant MOBP‐81 cDNA was determined and compared with tippy and ducky MOBP‐81. Wild‐type mouse MOBP‐81 protein was 99% identical to the rat homologue, and tippy and ducky MOBP‐81 were identical to the wild‐type sequence. Our results suggest that alterations in the Mobp gene are not the cause for the severe neurological phenotypes of ducky and tippy mice.
Subcellular Distribution of Glyceraldehyde‐3‐Phosphate Dehydrogenase in Cerebellar Granule Cells Undergoing Cytosine Arabinoside‐Induced Apoptosis
Authors: Saunders P. A. , Chalecka‐Franaszek E. , Chuang D.‐M.
Published: July 2010

Abstract: We have previously shown that cytosine arabinoside (AraC)‐induced apoptosis of cerebellar granule cells (CGCs) results in an increase of a 38‐kDa band on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, identified as glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH; EC 1.2.1.12). Antisense oligonucleotides to GAPDH mRNA afford acutely plated CGCs significant protection against AraC‐induced apoptosis. We used differential centrifugation to examine which subcellular components are affected. Treated and untreated cells were sonicated in 0.32 M sucrose and sequentially centrifuged at 1,000, 20,000, and 200,000 g, to obtain crude nuclear, mitochondrial, microsomal, and cytosolic fractions. Western blotting showed that the levels of GAPDH protein were markedly increased in the 1,000‐ and 20,000‐g pellets. The levels in the cytosolic supernatant were decreased dramatically by AraC in acutely plated CGCs but not in cells 24 h after plating. It is noteworthy that although GAPDH protein in the pellet fractions increased, the dehydrogenase activity of GAPDH decreased. Two other dehydrogenases, lactate dehydrogenase (EC 1.1.1.27) and glucose‐6‐phosphate dehydrogenase (EC 1.1.1.49), were not similarly affected, suggesting that the effect was GAPDH specific. These observations suggest that GAPDH levels change in specific organelles during apoptosis for reasons that are separate from its function as a glycolytic enzyme. The accumulation of GAPDH protein in specific subcellular loci may play a role in neuronal apoptosis.
Molecular Characterization and Differential Gene Induction of the Neuroendocrine‐Specific Genes Neurotensin, Neurotensin Receptor, PC1, PC2, and 7B2 in the Human Ocular Ciliary Epithelium
Authors: Ortego Javier , Coca‐Prados Miguel
Published: July 2010

Abstract: The ocular ciliary epithelium is a bilayer of neuroepithelial cells specialized in the secretion of aqueous humor fluid and the regulation of intraocular pressure. In this study, we report on the expression of the regulatory peptide neurotensin (NT) and a set of differentiated neuroendocrine markers including neurotensin receptors (NTrs), the prohormone convertases furin, PC1, and PC2, and the neuroendocrine polypeptide 7B2 in the ciliary epithelium. Using a human cell line, ODM‐2, derived from the nonpigmented ciliary epithelium, we demonstrate that (1) NT expression is highly activated by nerve growth factor, glucocorticoid, and activators of adenylate cyclase; (2) NTr expression is up‐regulated by selective ligand‐activated β2‐adrenergic receptor; and (3) PC1 and PC2 expression are up‐regulated via distinct signaling transduction pathways. PC1 gene expression is activated by phorbol ester, and PC2 by the same inducers as those of NT expression. A radioimmunoassay for NT detected an NT‐like immunoreactivity in human ciliary epithelium and ODM‐2 cell extracts, in aqueous humor, and in conditioned culture medium. The results support the view that the entire ciliary epithelium functions as a neuroendocrine tissue, synthesizing, processing, and releasing NT into the aqueous humor where it may exert important physiological functions through autocrine and/or paracrine mechanisms.
Expression of Neuronal Kinesin Heavy Chain Is Developmentally Regulated in the Central Nervous System of the Rat
Authors: Vignali G. , Lizier C. , Sprocati M. T. , Sirtori C. , Battaglia G. , Navone F.
Published: July 2010

Abstract: The kinesin family of motor proteins comprises at least two isoforms of conventional kinesin encoded by different genes: ubiquitous kinesin, expressed in all cells and tissues, and neuronal kinesin, expressed exclusively in neuronal cells. In the present study, we have analyzed the expression of the two kinesin isoforms by immunochemistry at different stages of development of the rat CNS. We have found that the level of expression of neuronal kinesin is five to eight times higher in developing than in adult rat brains, whereas that of ubiquitous kinesin is only ∼2.5 times higher in maturing versus adult brains. Moreover, we have studied the distribution of neuronal kinesin by light microscopic immunocytochemistry in the rat brain at different postnatal ages and have found this protein not only to be more highly expressed in juvenile than in adult rat brains but also to show a different pattern of distribution. In particular, tracts of axonal fibers were clearly stained at early postnatal stages of development but were markedly unlabeled in adult rat brains. Our results indicate that the expression of at least one isoform of conventional neuron‐specific kinesin is up‐regulated in the developing rat CNS and suggest that this protein might play an important role in microtubule‐based transport during the maturation of neuronal cells in vivo.
Neonatal Oligodendrocytes Contain and Secrete Neuregulins In Vitro
Authors: Raabe Timothy D. , Clive Diana R. , Wen Duanzhi , DeVries George H.
Published: July 2010

Abstract: The factors that influence the development of oligodendrocyte (OLG) progenitors into mature OLGs remain elusive. Recent evidence has suggested that neu differentiation factor (NDF), which is a member of the neuregulin family of growth factors, influences the development of glial cells, including Schwann cells, astrocytes, and OLGs. Neurons are postulated to be the source of neuregulins, because neurons closely interact with these glial cells during development. In this report, we have identified the mRNA for both isoform families of NDF in cultured neonatal (immature) OLGs. We have also demonstrated that cultured neonatal OLGs contain and secrete NDF protein. These data raise the possibility that NDF could be used in an autocrine/paracrine loop by neonatal OLGs during development for survival, proliferation, and/or differentiation.
Phorbol Ester and Calcium Regulation of Corticotrophin‐Releasing Factor Receptor 1 Expression in a Neuronal Cell Line
Authors: Iredale Philip A. , Bundey Richard , Duman Ronald S.
Published: July 2010

Abstract: We have previously demonstrated that corticotrophin‐releasing factor receptor 1 (CRF‐R1) mRNA levels can be down‐regulated via activation of the cyclic AMP pathway in CATH.a cells, a neuronal cell line. In this study, we show evidence for down‐regulation of CRF‐R1 mRNA levels via activation of the protein kinase C (PKC) and calcium second messenger pathways. Incubation of CATH.a cells with phorbol 12‐myristate 13‐acetate (PMA), an activator of PKC, resulted in a time‐ and concentration‐dependent down‐regulation of CRF‐R1 mRNA levels. Pretreatment with the inactive phorbol ester 4α‐phorbol failed to influence significantly CRF‐R1 mRNA levels. Incubation with carbachol, a cholinergic agonist known to activate PKC and increase intracellular calcium levels via phosphatidylinositol breakdown, also down‐regulated CRF‐R1 mRNA levels. Intracellular calcium levels were directly increased using A23187, a calcium ionophore, and thapsigargin, a calcium‐ATPase inhibitor. Elevation of intracellular calcium content using either A23187 or thapsigargin significantly down‐regulated levels of CRF‐R1 mRNA. Furthermore, chelation of calcium with EGTA or blockade of voltage‐dependent calcium channels with nifedipine inhibited agonist‐mediated down‐regulation of CRF‐R1 mRNA levels. These results indicate that activation of PKC or calcium signal transduction pathways is sufficient to cause down‐regulation of CRF‐R1 mRNA levels and that calcium is required for agonist‐mediated down‐regulation of this receptor.
Synaptic Vesicle Recycling in Cultured Cerebellar Granule Cells: Role of Vesicular Acidification and Refilling
Authors: Cousin Michael A. , Nicholls David G.
Published: July 2010

Abstract: The role of the transvesicular protonmotive force in synaptic vesicle recycling was investigated in cultured cerebellar granule cells. The vesicular V‐ATPase was inhibited by 1 µM bafilomycin A1; as an alternative, the pH component of the gradient was selectively collapsed by equilibration of the cells with 10 mM methylamine and monitored with the fluorescent probe Lysosensor Green. Electrical field‐evoked exocytosis of d‐[3H]aspartate was inhibited by bafilomycin A1 but not by methylamine, indicating that a transvesicular membrane potential rather than pH gradient is required for transmitter retention within vesicles. In contrast, neither compound affected the field‐evoked uptake, recycling, or destaining of the vesicle‐specific dye FM2‐10; thus, vesicles whose lumens were neutral and/or depleted of transmitter could still recycle in the nerve terminal. No exhaustion of d‐[3H]aspartate exocytosis was observed when cells were subjected to six consecutive trains of field stimuli (40 Hz/10 s separated by 10 s). In contrast, the release of preloaded FM2‐10 was reduced by ∼50%, with each stimulus indicating that unlabeled vesicles with accumulated d‐[3H]aspartate were competing with labeled vesicles for exocytosis. As d‐[3H]aspartate was accumulated rapidly across the vesicle membrane from the large cytoplasmic pool, the transmitter‐loaded but unlabelled vesicles may represent refilled recycling vesicles. FM2‐10 destaining and d‐[3H]aspartate exocytosis were reduced in parallel at low frequencies, challenging a role for transient vesicle fusion.
Dexamethasone Prevents Hypoxia/Ischemia‐Induced Reductions in Cerebral Glucose Utilization and High‐Energy Phosphate Metabolites in Immature Brain
Authors: Tuor U. I. , Yager J. Y. , Bascaramurty S. , Del Bigio M. R.
Published: July 2010

Abstract: We examined the potential importance of dexamethasone‐mediated alterations in energy metabolism in providing protection against hypoxic‐ischemic brain damage in immature rats. Seven‐day‐old rats (n = 165) that had been treated with dexamethasone (0.1 mg/kg, i.p.) or vehicle were assigned to control or hypoxic‐ischemic groups (unilateral carotid artery occlusion plus 2–3 h of 8% oxygen at normothermia). The systemic availability of alternate fuels such as β‐hydroxybutyrate, lactate, pyruvate, and free fatty acids was not altered by dexamethasone treatment, and, except for glucose, brain levels were also unaffected. At the end of hypoxia, levels of cerebral high‐energy phosphates (ATP and phosphocreatine) were decreased in vehicle‐ but relatively preserved in dexamethasone‐treated animals. The local cerebral metabolic rate of glucose utilization (lCMRgl) was decreased modestly under control conditions in dexamethasone‐treated animals, whereas cerebral energy use measured in a model of decapitation ischemia did not differ significantly between groups. The lCMRgl increased markedly during hypoxia‐ischemia (p < 0.05) and remained elevated throughout ischemia in dexamethasone‐but not vehicle‐treated groups, indicating an enhanced glycolytic flux with dexamethasone treatment. Thus, dexamethasone likely provides protection against hypoxic‐ischemic damage in immature rats by preserving cerebral ATP secondary to a maintenance of glycolytic flux.
Evidence of Increased Oxidative Damage in Both Sporadic and Familial Amyotrophic Lateral Sclerosis
Authors: Ferrante Robert J. , Browne Susan E. , Shinobu Leslie A. , Bowling Allen C. , Baik M. Jay , MacGarvey Usha , Kowall Neil W. , Brown Robert H. , Beal M. Flint
Published: July 2010

Abstract: Some cases of autosomal dominant familial amyotrophic lateral sclerosis (FALS) are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1), suggesting that oxidative damage may play a role in ALS pathogenesis. To further investigate the biochemical features of FALS and sporadic ALS (SALS), we examined markers of oxidative damage to protein, lipids, and DNA in motor cortex (Brodmann area 4), parietal cortex (Brodmann area 40), and cerebellum from control subjects, FALS patients with and without known SOD mutations, SALS patients, and disease controls (Pick's disease, progressive supranuclear palsy, diffuse Lewy body disease). Protein carbonyl and nuclear DNA 8‐hydroxy‐2′‐deoxyguanosine (OH8dG) levels were increased in SALS motor cortex but not in FALS patients. Malondialdehyde levels showed no significant changes. Immunohistochemical studies showed increased neuronal staining for hemeoxygenase‐1, malondialdehyde‐modified protein, and OH8dG in both SALS and FALS spinal cord. These studies therefore provide further evidence that oxidative damage may play a role in the pathogenesis of neuronal degeneration in both SALS and FALS.
Regulation of Spontaneous Activity of the δ‐Opioid Receptor: Studies of Inverse Agonism in Intact Cells
Authors: Merkouris Manolis , Mullaney Ian , Georgoussi Zafiroula , Milligan Graeme
Published: July 2010

Abstract: Adenylyl cyclase activity was measured following labelling of the cellular ATP pool with [3H]adenine in intact Rat‐1 fibroblasts that had been stably transfected to express the murine δ‐opioid receptor (clone D2). Basal [3H]cyclic AMP accumulation was low and was increased substantially by the addition of the diterpene forskolin. The synthetic enkephalin d‐Ala2,d‐Leu5 enkephalin (DADLE) produced strong inhibition of forskolin‐amplified [3H]cyclic AMP production, whereas the δ‐opioid ligand ICI174864 augmented forskolin‐amplified adenylyl cyclase activity. Naloxone was unable to mimic the effects of ICI174864, and coincubation of the cells with these two ligands attenuated the effect of ICI174864. The EC50 (9.4 ± 0.6 × 10−8M) for ICI174864 augmentation of forskolin‐stimulated adenylyl cyclase was equal to its estimated Ki. Pertussis toxin pretreatment of clone D2 cells prevented both this effect of ICI174864 and the inhibition produced by DADLE. Use of a Cytosensor microphysiometer demonstrated that treatment of clone D2 cells with DADLE increased and that with ICI174864 decreased the basal rate of cellular proton extrusion. By using these two distinct experimental strategies, ICI174864 was shown to function in a manner anticipated for an inverse agonist, demonstrating that such effects can be observed in intact cells and are not restricted to assays performed on membrane preparations.
Transient Expression of PG‐M/Versican, a Large Chondroitin Sulfate Proteoglycan in Developing Chicken Retina
Authors: Zako Masahiro , Shinomura Tamayuki , Miyaishi Osamu , Iwaki Masayoshi , Kimata Koji
Published: July 2010

Abstract: We previously showed the expression of PG‐M/versican in embryonic chicken retina. In this study, we characterized the alternatively spliced forms of PG‐M/versican and their developmental regulation to investigate the implication of PG‐M/versican in neurite outgrowth from retinal cells during development. On day 5, the immunolocalization of PG‐M was first observed at the inner surface of neural retina. On day 7, the pronounced staining was observed in the nerve fiber layer and inner plexiform layer where neural networks of ganglion cells were being formed. As the development proceeded, more intensive staining was observed in these layers. The staining peaked on day 14 and then decreased. Northern analysis and western blotting revealed the presence of a single‐sized transcript (13 kb) and the PG‐M/versican core protein (550 kDa) on day 14, but the absence of any transcripts or protein bands on day 20, indicating a transient expression of PG‐M+ (VO), the alternatively spliced form with the most abundant sites for the chondroitin sulfate attachment. Taken together, it is likely that PG‐M/versican is involved in neurite outgrowth from ganglion cells during retinal development, and antiadhesion activity of its chondroitin sulfate chains may be important for regulation.
The Amyloid Precursor Protein Is Not Enriched in Caveolae‐Like, Detergent‐Insoluble Membrane Microdomains
Authors: Parkin Edward T. , Hussain Ishrut , Turner Anthony J. , Hooper Nigel M.
Published: July 2010

Abstract: The amyloid precursor protein may be processed by several different pathways, one of which produces the amyloid β‐peptide βA4 present in the amyloid plaques characteristic of Alzheimer's disease. A recent report suggested that axonal‐amyloid precursor protein is present in a membrane fraction “with caveolae‐like properties.” In the present study we have isolated detergent‐insoluble, caveolae‐like membranes from both mouse cerebellum and the human neuroblastoma cell line SH‐SY5Y. Detergent‐insoluble membranes from mouse cerebellum retained nearly all of the glycosylphosphatidylinositol‐anchored proteins—alkaline phosphatase, 5′‐nucleotidase, and the F3 protein—while excluding the majority of the plasmalemmal marker protein alkaline phosphodiesterase I. Although the inositol trisphosphate receptor was highly enriched in this detergent‐insoluble fraction, neither amyloid precursor protein nor clathrin immunoreactivity could be detected. Similar results were obtained with SH‐SY5Y cells, where 5′‐nucleotidase activity was enriched at least 30‐fold in the detergent‐insoluble membranes, but no amyloid precursor protein or clathrin immunoreactivity could be detected. Caveolin could not be detected in microsomal membranes from either mouse cerebellum or SH‐SY5Y cells. These observations suggest that amyloid precursor protein is not normally present in detergent‐insoluble, caveolae‐like membrane microdomains.
Involvement of L‐Type‐Like Amino Acid Transporters in S‐Nitrosocysteine‐Stimulated Noradrenaline Release in the Rat Hippocampus
Authors: Satoh Souichi , Kimura Tatsuo , Toda Masahiro , Maekawa Mutuko , Ono Satoshi , Narita Hirokazu , Miyazaki Hiroyuki , Murayama Toshihiko , Nomura Yasuyuki
Published: July 2010

Abstract: Nitrogen oxides, such as nitric oxide, have been shown to regulate neuronal functions, including neurotransmitter release. We investigated the effect of S‐nitroso‐l‐cysteine (SNC) on noradrenaline (NA) release in the rat hippocampus in vivo and in vitro. SNC stimulated [3H]NA release from prelabeled hippocampal slices in a dose‐dependent manner. SNC stimulated endogenous NA release within 30 min to almost five times the basal level in vivo (microdialysis in freely moving rats). In a Na+‐containing Tyrode's buffer, SNC‐stimulated [3H]NA release was inhibited 30% by the coaddition of l‐leucine. In the Na+‐free, choline‐containing buffer, SNC‐stimulated [3H]NA release, which was similar to that in the Na+‐containing buffer, was inhibited markedly by l‐leucine, l‐alanine, l‐methionine, l‐phenylalanine, and l‐tyrosine. The effects of the other amino acids examined were smaller or very limited. The effect of l‐leucine was stronger than that of d‐leucine. A specific inhibitor of the L‐type amino acid transporter, 2‐aminobicyclo[2.2.1]‐heptane‐2‐carboxylate (BCH), inhibited the effects of SNC on [3H]NA release in the Na+‐free buffer. Uptake of l‐[3H]leucine into the slices in the Na+‐free buffer was inhibited by SNC, BCH, and l‐phenylalanine, but not by l‐lysine. The effect of SNC on cyclic GMP accumulation was not inhibited by l‐leucine, although SNC stimulated cyclic GMP accumulation at concentrations up to 25 µM, much less than the concentration that stimulates NA release. These findings suggest that SNC is incorporated into rat hippocampus via the L‐type‐like amino acid transporter, at least in Na+‐free conditions, and that SNC stimulates NA release in vivo and in vitro in a cyclic GMP‐independent manner.
Phosphorylation of the Casein Kinase II Domain of B‐50 (GAP‐43) in Rat Cortical Growth Cones
Authors: Edgar Michael A. N. , Pasinelli Piera , DeWit M. , Anton Brian , Dokas Linda A. , Pastorino Lucia , DiLuca Monica , Cattabeni Flaminio , Gispen Willem H. , De Graan Pierre N. E.
Published: July 2010

Abstract: Growth‐associated phosphoprotein B‐50 is a neural protein kinase C (PKC) substrate enriched in nerve growth cones that has been implicated in growth cone plasticity. Here we investigated whether B‐50 is a physiological substrate for casein kinase II (CKII) in purified rat cortical growth cone preparations. Using site‐specific proteolysis and known modulators of PKC, in combination with immunoprecipitation, mass spectrometry, and phosphoamino acid analysis, we demonstrate that endogenous growth cone B‐50 is phosphorylated at multiple sites, on both serine and threonine residues. Consistent with previous reports, stimulation of PKC activity increased the phosphorylation of only those proteolytic fragments containing Ser41. Under basal conditions, however, phosphorylation was predominantly associated with fragments not containing Ser41. Mass spectrometry of tryptic digests of B‐50, which had been immunoprecipitated from untreated growth cones, revealed that in situ phosphorylation occurs within peptides B‐50181–198 and B‐5082–98. These peptides contain the major and minor in vitro CKII phosphosites, respectively. In addition, cyanogen bromide digestion of immunoprecipitated chick B‐50 generated a 4‐kDa C‐terminal B‐50 phosphopeptide, confirming that phosphorylation of the CKII domain occurs across evolutionary diverse species. We conclude that B‐50 in growth cones is not only a substrate for PKC, but also for CKII.
The Caenorhabditis elegans Avermectin Resistance and Anesthetic Response Gene unc‐9 Encodes a Member of a Protein Family Implicated in Electrical Coupling of Excitable Cells
Authors: Barnes T. M. , Hekimi S.
Published: July 2010

Abstract: Mutations in the unc‐9 gene of the nematode Caenorhabditis elegans cause abnormal forward locomotion and an egg‐retention phenotype. unc‐9 mutations also reduce the worms' sensitivity to avermectin and block a form of hypersensitivity to volatile anesthetics. We report here the cloning and molecular characterization of unc‐9 and show that it encodes a member of the OPUS family of proteins that is 56% identical to another OPUS protein, UNC‐7. It is significant that unc‐9 mutants share all phenotypes with unc‐7 mutants. Mutants in another gene, unc‐124, also share all tested phenotypes with unc‐9 mutants, including identical locomotory and egg‐laying defects, suggesting that multiple genes are required for the same biochemical function. OPUS proteins are implicated in the function of invertebrate gap junctions, and, based on a new alignment including 24 members from C. elegans, we present a refined model for the structure of OPUS proteins suggesting that oligomers could form a hydrophilic pore. We also show that alteration of highly conserved proline residues in UNC‐9 leads to a cold sensitivity that likely affects a step in protein expression rather than function. Finally, we speculate on the basis of the avermectin resistance and anesthetic response phenotypes.
Brain CRMP Forms Heterotetramers Similar to Liver Dihydropyrimidinase
Authors: Wang Li‐Hsien , Strittmatter Stephen M.
Published: July 2010

Abstract: The cytoplasmic collapsin response mediator protein CRMP62 is involved in the signaling cascade initiated by collapsin‐1, which collapses neuronal growth cones. To investigate the mechanism of CRMP action, we screened mouse and human fetal cDNA libraries by the yeast two‐hybrid method with CRMP as bait. Clones encoding CRMP1 and CRMP4 were isolated, suggesting that the CRMPs form multimers. This finding was confirmed by expressing various rat CRMP cDNAs in the yeast two‐hybrid system. Rat CRMP isoforms show differential association with one another. Heterooligomerization is preferred in both two‐hybrid and in vitro binding assays. Purified bovine brain CRMP migrates as a tetramer during size exclusion chromatography. Examination of binding with truncated forms of CRMPs indicates that the avid association of CRMPs requires nearly intact proteins. Through the analysis of CRMP chimeras, CRMP amino acids 8–134 and 281–435 are found to be essential for CRMP oligomerization. The tetrameric structure of CRMP resembles that of liver dihydropyrimidinase (DHPase), a protein that shares sequence similarity with the CRMPs. Although purified brain CRMP does not hydrolyze several DHPase substrates, it is likely that a related activity accounts for CRMP participation in neuronal growth cone signaling.