bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Calcineurin in Ca2+‐Induced Release of Catecholamines and Neuropeptides
Authors: Hens Jacques J. H. , De Wit Marina , Ghijsen Wim E. J. M. , Leenders A. G. Miriam , Boddeke Hendrik W. G. M. , Kissmehl Roland , Wiegant Victor M. , Weller Ulrich , Gispen Willem‐Hendrik , De Graan Pierre N. E.
Published: July 2010

Abstract: Neurotransmission requires rapid docking, fusion, and recycling of neurotransmitter vesicles. Several of the proteins involved in this complex Ca2+‐regulated mechanism have been identified as substrates for protein kinases and phosphatases, e.g., the synapsins, synaptotagmin, rabphilin3A, synaptobrevin, munc18, MARCKS, dynamin I, and B‐50/GAP‐43. So far most attention has focused on the role of kinases in the release processes, but recent evidence indicates that phosphatases may be as important. Therefore, we investigated the role of the Ca2+/calmodulin‐dependent protein phosphatase calcineurin in exocytosis and subsequent vesicle recycling. Calcineurin‐neutralizing antibodies, which blocked dynamin I dephosphorylation by endogenous synaptosomal calcineurin activity, but had no effect on the activity of protein phosphatases 1 or 2A, were introduced into rat permeabilized nerve terminals and inhibited Ca2+‐induced release of [3H]noradrenaline and neuropeptide cholecystokinin‐8 in a specific and concentration‐dependent manner. Our data show that the Ca2+/calmodulin‐dependent phosphatase calcineurin plays an essential role in exocytosis and/or vesicle recycling of noradrenaline and cholecystokinin‐8, transmitters stored in large dense‐cored vesicles.
Glia Modulate NMDA‐Mediated Signaling in Primary Cultures of Cerebellar Granule Cells
Authors: Beaman‐Hall Carol M. , Leahy J. Clancy , Benmansour Saloua , Vallano Mary Lou
Published: July 2010

Abstract: Excessive activation of N‐methyl‐d‐aspartate (NMDA) receptor channels (NRs) is a major cause of neuronal death associated with stroke and ischemia. Cerebellar granule neurons in vivo, but not in culture, are relatively resistant to toxicity, possibly owing to protective effects of glia. To evaluate whether NR‐mediated signaling is modulated when developing neurons are cocultured with glia, the neurotoxic responses of rat cerebellar granule cells to applied NMDA or glutamate were compared in astrocyte‐rich and astrocyte‐poor cultures. In astrocyte‐poor cultures, significant neurotoxicity was observed in response to NMDA or glutamate and was inhibited by an NR antagonist. Astrocyte‐rich neuronal cultures demonstrated three significant differences, compared with astrocyte‐poor cultures: (a) Neuronal viability was increased; (b) glutamate‐mediated neurotoxicity was decreased, consistent with the presence of a sodium‐coupled glutamate transport system in astrocytes; and (c) NMDA‐ but not kainate‐mediated neurotoxicity was decreased, in a manner that depended on the relative abundance of glia in the culture. Because glia do not express NRs or an NMDA transport system, the mechanism of protection is distinct from that observed in response to glutamate. No differences in NR subunit composition (evaluated using RT‐PCR assays for NR1 and NR2 subunit mRNAs), NR sensitivity (evaluated by measuring NR‐mediated changes in intracellular Ca2+ levels), or glycine availability as a coagonist (evaluated in the presence and absence of exogenous glycine) were observed between astrocyte‐rich and astrocyte‐poor cultures, suggesting that glia do not directly modulate NR composition or function. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, blocked NMDA‐mediated toxicity in astrocyte‐poor cultures, raising the possibility that glia effectively reduce the accumulation of highly diffusible and toxic arachidonic acid metabolites in neurons. Alternatively, glia may alter neuronal development/phenotype in a manner that selectively reduces susceptibility to NR‐mediated toxicity.
Nonsteroidal Anti‐Inflammatory Drugs Increase Tumor Necrosis Factor Production in the Periphery but Not in the Central Nervous System in Mice and Rats
Authors: Sacco Silvano , Agnello Davide , Sottocorno Marcello , Lozza Gianluca , Monopoli Angela , Villa Pia , Ghezzi Pietro
Published: July 2010

Abstract: Nonsteroidal anti‐inflammatory drugs (NSAIDs), which inhibit prostaglandin (PG) synthesis, augment production of tumor necrosis factor (TNF) in most experimental models. We investigated the effect of two NSAIDs, indomethacin and ibuprofen, on the production of TNF in the CNS induced by intracerebroventricular injection of lipopolysaccharide (LPS). Indomethacin and ibuprofen, administered intraperitoneally, augmented (three‐ to ninefold) the levels of TNF in serum and peripheral organs of mice injected intraperitoneally with LPS and in rats with adjuvant arthritis (up to a sevenfold increase). However, NSAIDs (intraperitoneally or intracerebroventricularly) did not increase brain TNF production induced by intravenous LPS. In fact, indomethacin decreased (1.4–1.8‐fold) TNF levels in the spinal cord of rats with experimental autoimmune encephalomyelitis and in the cortex of rats with focal cerebral ischemia. Systemic administration of iloprost inhibited serum TNF levels after intraperitoneal LPS, whereas intracerebroventricular injection of iloprost or PGE2 did not inhibit brain TNF induced by intracerebroventricular LPS. Both peripheral and central TNF productions were inhibited by cyclic AMP level‐elevating agents or dexamethasone. Thus, a PG‐driven negative feedback controls TNF production in the periphery but not in the CNS.
Prenatal Ethanol Exposure Decreases GAP‐43 Phosphorylation and Protein Kinase C Activity in the Hippocampus of Adult Rat Offspring
Authors: Perrone‐Bizzozero Nora I. , Isaacson Tove V. , Keidan Gregory M. O. , Eriqat Cheryl , Meiri Karina F. , Savage Daniel D. , Allan Andrea M.
Published: July 2010

Abstract: Consumption of moderate quantities of ethanol during pregnancy produces deficits in long‐term potentiation in the hippocampal formation of adult offspring. Protein kinase C (PKC)‐mediated phosphorylation of the presynaptic protein GAP‐43 is critical for the induction of long‐term potentiation. We tested the hypothesis that this system is affected in fetal alcohol‐exposed (FAE) rats by measuring GAP‐43 phosphorylation and PKC activity in the hippocampus of adult offspring of rat dams that had consumed one of three diets throughout gestation: (a) a 5% ethanol liquid diet, which produced a maternal blood ethanol concentration of 83 mg/dl (FAE); (b) an isocalorically equivalent 0% ethanol diet (pair‐fed); or (c) lab chow ad libitum. Western blot analysis using specific antibodies to PKC‐phosphorylated GAP‐43 revealed that FAE rats had an ∼50% reduction in the proportion of phosphorylated GAP‐43. Similarly, we found that PKC‐mediated incorporation of 32P into GAP‐43 was reduced by 85% in hippocampal slices from FAE rats compared with both control groups. FAE animals also showed a 50% reduction in total hippocampal PKC activity, whereas the levels of six major PKC isozymes did not change in any of the diet groups. These results suggest that GAP‐43 phosphorylation deficits in rats prenatally exposed to moderate levels of ethanol are not due to alterations in the expression of either the enzyme or substrate protein, but rather to a defect in kinase activation.
Conjugates of Catecholamines with Cysteine and GSH in Parkinson's Disease: Possible Mechanisms of Formation Involving Reactive Oxygen Species
Authors: Spencer Jeremy P. E. , Jenner Peter , Daniel Susan E. , Lees Andrew J. , Marsden David C. , Halliwell Barry
Published: July 2010

Abstract: Oxidation of l‐3,4‐dihydroxyphenylalanine (l‐DOPA) and dopamine (DA) to generate semiquinones/quinones, oxygen radicals, and other reactive oxygen species may play a role in neuronal cell death in Parkinson's disease (PD). In particular, semiquinones/quinones can form conjugates with thiol compounds such as GSH and cysteine. Exposure of l‐DOPA, DA, and other catecholamines to a system generating O2•− radical led to O2•−‐dependent depletion of added GSH (or cysteine), accompanied by the formation of thiol‐DA or ‐DOPA adducts as detected by HPLC. Superoxide could additionally cause destruction of these adducts. Iron or copper ions could also promote conjugate formation between GSH or cysteine and DA and l‐DOPA, especially if H2O2 was present. We applied HPLC to measure glutathionyl and cysteinyl conjugates of l‐DOPA, DA, and 3,4‐dihydroxyphenylacetic acid (DOPAC) in postmortem brain samples from PD patients and normal control subjects. Conjugates were detected in most brain areas examined, but levels were highest in the substantia nigra and putamen. In most regions, adduct levels were lower in PD, but there were significant increases in cysteinyl adducts of l‐DOPA, DA, and DOPAC in PD substantia nigra, suggesting that acceleration of l‐DOPA/DA oxidation occurs in PD, although we cannot say if this is a primary feature of the disease or if it is related to therapy with l‐DOPA. In vitro, conjugate formation could be inhibited by the dithiol dihydrolipoate but not by its oxidised form, lipoic acid.
Alzheimer's β‐Amyloid Peptide 1–42 Induces a Phagocytic Response in Murine Microglia
Authors: Kopec Karla K. , Carroll Richard T.
Published: July 2010

Abstract: β‐Amyloid (Aβ) peptides are a key component of the senile plaques that characterize Alzheimer's disease. Cytokine‐producing microglia have been shown to be intimately associated with amyloid deposits and have also been implicated as scavengers responsible for clearing Aβ deposits. However, little is known about the initial activation of these microglia or the effect of Aβ on phagocytosis. Murine BV‐2 microglia were used to assess the effect of synthetic Aβ 1–42 on phagocytosis by quantifying uptake of fluorescent microspheres, acetylated low‐density lipoproteins, and zymosan particles by flow cytometry. Aβ 1–42 stimulated microglial phagocytosis in a time‐ and dose‐dependent manner. Aβ fibrils produced the greatest potentiation, and once activated, phagocytosis remained elevated after removal of Aβ from the cultures. Aβ‐stimulated phagocytosis could be blocked if proteoglycans were first complexed to Aβ fibrils. These data suggest that Aβ fibrils act as an immune signal to stimulate microglial phagocytosis and that extracellular matrix molecules may modify Aβ function.
Effect of Serine and Ethanolamine Administration on Phospholipid‐Related Compounds and Neurotransmitter Amino Acids in the Rabbit Hippocampus
Authors: Buratta Sandra , Hamberger Anders , Ryberg Henrik , Nyström Britta , Sandberg Mats , Mozzi Rita
Published: July 2010

Abstract: The report concerns mechanisms for the increase of extracellular levels of ethanolamine and phosphoethanolamine in CNS regions, such as the hippocampus, in transient brain ischemia, hypoglycemia, seizures, etc. l‐Serine (2.5–10 mM), d‐serine (10 mM), or ethanolamine (10 mM) was administered for 20 min via a microdialysis tubing to the hippocampus of unanesthetized rabbits. The concentrations of primary amines were determined in the dialysates. When levels were elevated 10–100 times in the extracellular fluid, l‐serine caused a dose‐dependent increase of the concentration of extracellular ethanolamine. Ethanolamine caused a corresponding, although somewhat smaller, increase in serine levels. Furthermore, l‐serine also induced an increased concentration of phosphoethanolamine that was delayed in time relative to the peak of ethanolamine. d‐Serine was as effective as l‐serine in raising ethanolamine levels but had no effect on phosphoethanolamine. Ethanolamine, but not l‐serine, also increased extracellular glutamate/aspartate levels in an MK‐801‐dependent fashion. A similar effect, but delayed in time, was observed with d‐serine. These effects were inhibited by MK‐801. The concentrations of other amino acids were not significantly affected. The characteristics of the effects are suggestive of base exchange reactions between serine and ethanolamine and between ethanolamine and serine glycerophospholipids, respectively, in neuronal plasma membranes.
Zinc Potentiation of the Glycine Receptor Chloride Channel Is Mediated by Allosteric Pathways
Authors: Lynch Joseph W. , Jacques Patrice , Pierce Kerrie D. , Schofield Peter R.
Published: July 2010

Abstract: Molecular mechanisms of zinc potentiation were investigated in recombinant human α1 glycine receptors (GlyRs) by whole‐cell patch‐clamp recording and [3H]strychnine binding assays. In the wild‐type (WT) GlyR, 1 µM zinc enhanced the apparent binding affinity of the agonists glycine and taurine and reduced their concentrations required for half‐maximal activation. Thus, in the WT GlyR, zinc potentiation apparently occurs by enhancing agonist binding. However, analysis of GlyRs incorporating mutations in the membrane‐spanning domain M1–M2 and M2–M3 loops, which are both components of the agonist gating mechanism, indicates that most mutations uncoupled zinc potentiation from glycine‐gated currents but preserved zinc potentiation of taurine‐gated currents. One such mutation in the M2–M3 loop, L274A, abolished the ability of zinc to potentiate taurine binding but did not inhibit zinc potentiation of taurine‐gated currents. In this same mutant where taurine acts as a partial agonist, zinc potentiated taurine‐gated currents but did not potentiate taurine antagonism of glycine‐gated currents, suggesting that zinc interacts selectively with the agonist transduction pathway. The intracellular M246A mutation, which is unlikely to bind zinc, also disrupted zinc potentiation of glycine currents. Thus, zinc potentiation of the GlyR is mediated via allosteric mechanisms that are independent of its effects on agonist binding.
Identification of Residues in Transmembrane Regions III and VI that Contribute to the Ligand Binding Site of the Serotonin 5‐HT6 Receptor
Authors: Boess Frank G. , Monsma Frederick J. , Sleight Andrew J.
Published: July 2010

Abstract: We have examined the ligand binding site of the serotonin 5‐HT6 receptor using site‐directed mutagenesis. Replacing the highly conserved Asp106 in transmembrane region III by asparagine eliminated d‐[3H]lysergic acid diethylamide ([3H]LSD) binding to the mutant receptor transiently expressed in HEK293 cells. The potency of 5‐HT and LSD to stimulate adenylyl cyclase was reduced by 3,600‐ and 500‐fold, respectively, suggesting that an ionic interaction between the positively charged amino group of 5‐HT and D106 is essential for high‐affinity binding and important for receptor activation. In addition, basal cyclic AMP levels in cells expressing this mutant were increased. Mutation of a tryptophan residue one helix turn toward the extracellular side of transmembrane region III (Trp102) to phenylalanine produced significant changes in the binding affinity and potency of several ligands, consistent with a role of this residue in the formation of the ligand binding site. The exchange of two neighboring residues in the carboxy‐terminal half of transmembrane region VI (Ala287 and Asn288) for leucine and serine resulted in a mutant receptor with increased affinities (seven‐ to 30‐fold) for sumatriptan and several ergopeptine ligands. The identification of these interactions will help to improve models of the 5‐HT6 receptor ligand binding site.
Steroidogenic Acute Regulatory Protein (StAR) Transcripts Constitutively Expressed in the Adult Rat Central Nervous System: Colocalization of StAR, Cytochrome P‐450SCC (CYP XIA1), and 3β‐Hydroxysteroid Dehydrogenase in the Rat Brain
Authors: Furukawa Aizo , Miyatake Akira , Ohnishi Taira , Ichikawa Yoshiyuki
Published: July 2010

Abstract: Steroidogenic acute regulatory protein (StAR) is a 30‐kDa protein involved in the transport of cholesterol to the inner mitochondrial membrane and thus plays a key role in steroid biosynthesis. To clarify the implications of this protein in neurosteroid biosynthesis, we examined the possible expression of a StAR transcript in the adult rat CNS and detected it. cDNA cloning and sequencing analysis revealed that two forms of StAR mRNAs are expressed in the brain in the same manner as in the adrenal gland, indicating that they are fully functional and not minor gene transcripts. An RNase protection assay quantitatively revealed that the amount of the rat StAR transcript in brain was two to three orders of magnitude lower than that in the adrenal gland. An in situ hybridization study, involving antisense riboprobes, revealed that StAR transcripts were abundant in the cerebral cortex, hippocampus, dentate gyrus, olfactory bulb, cerebellar granular layer, and Purkinje cells. Furthermore, other steroidogenic enzymes, side‐chain cleavage cytochrome P‐450SCC (CYP XIA1) and 3β‐hydroxysteroid dehydrogenase/Δ5‐Δ4 isomerase (EC 1.1.1.145), were found to be coexpressed in the hippocampus, dentate gyrus, cerebellar granular layer, and Purkinje cells. These findings strongly indicate that neurosteroids are synthesized in a region‐specific manner in the brain.
Molecular Characterization and Expression of Cloned Human Galanin Receptors GALR2 and GALR3
Authors: Kolakowski Lee F. , O'Neill Gary P. , Howard Andrew D. , Broussard Suzanne R. , Sullivan Kathleen A. , Feighner Scott D. , Sawzdargo Marek , Nguyen Tuan , Kargman Stacia , Shiao Lin‐Lin , Hreniuk Donna L. , Tan Carina P. , Evans Jilly , Abramovitz Mark , Chateauneuf Anne , Coulombe Nathalie , Ng Gordon , Johnson Michael P. , Tharian Anita , Khoshbouei Habibeh , George Susan R. , Smith Roy G. , O'Dowd Brian F.
Published: July 2010

Abstract: Galanin is a 29‐ or 30‐amino acid peptide with wide‐ranging effects on hormone release, feeding behavior, smooth muscle contractility, and somatosensory neuronal function. Three distinct galanin receptor (GALR) subtypes, designated GALR1, 2, and 3, have been cloned from the rat. We report here the cloning of the human GALR2 and GALR3 genes, an initial characterization of their pharmacology with respect to radioligand binding and signal transduction pathways, and a profile of their expression in brain and peripheral tissues. Human GALR2 and GALR3 show, respectively, 92 and 89% amino acid sequence identity with their rat homologues. Radioligand binding studies with 125I‐galanin show that recombinant human GALR2 binds with high affinity to human galanin (KD = 0.3 nM). Human GALR3 binds galanin with less affinity (IC50 of 12 nM for porcine galanin and 75 nM for human galanin). Human GALR2 was shown to couple to phospholipase C and elevation of intracellular calcium levels as assessed by aequorin luminescence in HEK‐293 cells and by Xenopus melanophore pigment aggregation and dispersion assays, in contrast to human GALR1 and human GALR3, which signal predominantly through inhibition of adenylate cyclase. GALR2 mRNA shows a wide distribution in the brain (mammillary nuclei, dentate gyrus, cingulate gyrus, and posterior hypothalamic, supraoptic, and arcuate nuclei), and restricted peripheral tissue distribution with highest mRNA levels detected in human small intestine. In comparison, whereas GALR3 mRNA was expressed in many areas of the rat brain, there was abundant expression in the primary olfactory cortex, olfactory tubercle, the islands of Calleja, the hippocampal CA regions of Ammon's horn, and the dentate gyrus. GALR3 mRNA was highly expressed in human testis and was detectable in adrenal gland and pancreas. The genes for human GALR2 and 3 were localized to chromosomes 17q25 and 22q12.2–13.1, respectively.
Distinct Influx Pathways, Not Calcium Load, Determine Neuronal Vulnerability to Calcium Neurotoxicity
Authors: Sattler Rita , Charlton Milton P. , Hafner Mathias , Tymianski Michael
Published: July 2010

Abstract: Many forms of neurodegeneration are ascribed to excessive cellular Ca2+ loading (Ca2+ hypothesis). We examined quantitatively whether factors other than Ca2+ loading were determinants of excitotoxic neurodegeneration. Cell survival, morphology, free intracellular Ca2+ concentration ([Ca2+]i), and 45Ca2+ accumulation were measured in cultured cortical neurons loaded with known quantities of Ca2+ through distinct transmembrane pathways triggered by excitatory amino acids, cell membrane depolarization, or Ca2+ ionophores. Contrary to the Ca2+ hypothesis, the relationships between Ca2+ load and cell survival, free [Ca2+]i, and Ca2+‐induced morphological alterations depended primarily on the route of Ca2+ influx, not the Ca2+ load. Notably, Ca2+ loading via NMDA receptor channels was toxic, whereas identical Ca2+ loads incurred through voltage‐sensitive Ca2+ channels were completely innocuous. Furthermore, accounting quantitatively for Ca2+ loading via NMDA receptors uncovered a previously unreported component of l‐glutamate neurotoxicity apparently not mediated by ionotropic or metabotropic glutamate receptors. It was synergistic with toxicity attributable to glutamate‐evoked Ca2+ loading, and correlated with enhanced cellular ATP depletion. This previously unrecognized toxic action of glutamate constituted a chief excitotoxic mechanism under conditions producing submaximal Ca2+ loading. We conclude that (a) Ca2+ neurotoxicity is a function of the Ca2+ influx pathway, not Ca2+ load, and (b) glutamate toxicity may not be restricted to its actions on glutamate receptors.
Enrichment of Presenilin 1 Peptides in Neuronal Large Dense‐Core and Somatodendritic Clathrin‐Coated Vesicles
Authors: Efthimiopoulos Spiros , Floor Erik , Georgakopoulos Anastasios , Shioi Junichi , Cui Wen , Yasothornsrikul Sukkid , Hook Vivian Y. H. , Wisniewski Thomas , Buee Luc , Robakis Nikolaos K.
Published: July 2010

Abstract: Presenilin 1 is an integral membrane protein specifically cleaved to yield an N‐terminal and a C‐terminal fragment, both membrane‐associated. More than 40 presenilin 1 mutations have been linked to early‐onset familial Alzheimer disease, although the mechanism by which these mutations induce the Alzheimer disease neuropathology is not clear. Presenilin 1 is expressed predominantly in neurons, suggesting that the familial Alzheimer disease mutants may compromise or change the neuronal function(s) of the wild‐type protein. To elucidate the function of this protein, we studied its expression in neuronal vesicular systems using as models the chromaffin granules of the neuroendocrine chromaffin cells and the major categories of brain neuronal vesicles, including the small clear‐core synaptic vesicles, the large dense‐core vesicles, and the somatodendritic and nerve terminal clathrin‐coated vesicles. Both the N‐ and C‐terminal presenilin 1 proteolytic fragments were greatly enriched in chromaffin granule and neuronal large dense‐core vesicle membranes, indicating that these fragments are targeted to these vesicles and may regulate the large dense‐core vesicle‐mediated secretion of neuropeptides and neurotransmitters at synaptic sites. The presenilin 1 fragments were also enriched in the somatodendritic clathrin‐coated vesicle membranes, suggesting that they are targeted to the somatodendritic membrane, where they may regulate constitutive secretion and endocytosis. In contrast, these fragments were not enriched in the small clear‐core synaptic vesicle or in the nerve terminal clathrin‐coated vesicle membranes. Taken together, our data indicate that presenilin 1 proteolytic fragments are targeted to specific populations of neuronal vesicles where they may regulate vesicular function. Although full‐length presenilin 1 was present in crude homogenates, it was not detected in any of the vesicles studied, indicating that, unlike the presenilin fragments, full‐length protein may not have a vesicular function.
Calcium‐Sensitive Fluorescent Dyes Can Report Increases in Intracellular Free Zinc Concentration in Cultured Forebrain Neurons
Authors: Cheng Chialin , Reynolds Ian J.
Published: July 2010

Abstract: High concentrations of Zn2+ are found in presynaptic terminals of excitatory neurons in the CNS. Zn2+ can be released during synaptic activity and modulate postsynaptic receptors, but little is known about the possibility that Zn2+ may enter postsynaptic cells and produce dynamic changes in the intracellular Zn2+ concentration ([Zn2+]i). We used fura‐2 and magfura‐2 to detect the consequences of Zn2+ influx in cultured neurons under conditions that restrict changes in intracellular Ca2+ and Mg2+ concentrations. The resulting ratio changes for both dyes were reversed completely by the Zn2+ chelator, N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethylenediamine, indicating that these dyes are measuring changes in [Zn2+]i. We found that fura‐2 was useful in measuring small increases in [Zn2+]i associated with exposure to Zn2+ alone that may be mediated by a Na+/Ca2+ exchanger. Magfura‐2, which has a lower affinity for Zn2+, was more useful in measuring larger agonist‐stimulated increases in [Zn2+]i. The coapplication of 300 µM Zn2+ and 100 µM glutamate/10 µM glycine resulted in a [Zn2+]i increase that was ∼40–100 nM in magnitude and could be inhibited by the NMDA receptor antagonist, MK‐801 (30 µM), or extracellular Na+. This suggests that Zn2+ influx can occur through at least two different pathways, leading to varying increases in [Zn2+]i. These findings demonstrate the feasibility of measuring changes in [Zn2+]i in neurons.
Coordinate Expression of the Vesicular Acetylcholine Transporter and Choline Acetyltransferase Following Septohippocampal Pathway Lesions
Authors: Gilmor Michelle L. , Counts Scott E. , Wiley Ronald G. , Levey Allan I.
Published: July 2010

Abstract: The gene for the vesicular acetylcholine transporter (VAChT) was recently cloned and found to be located within a 5′ noncoding intron of the gene for choline acetyltransferase (ChAT). There appear to be several shared and unique promoters for each gene, suggesting that control of expression of these two genes can be either coordinated or independent. Two lesions, axotomy and immunotoxin, directed at the well defined septohippocampal cholinergic pathway were used to determine VAChT and ChAT protein expression in the degenerating terminal fields in the hippocampus and the cell bodies of the medial septum nucleus after injury. Two weeks after lesioning, decreases of up to 90% in VAChT were found in the affected hippocampus by immunoblotting and immunocytochemistry, similar to ChAT activity. The number of VAChT‐ and ChAT‐immunopositive neurons in the medial septum decreased by up to 95%. Eight weeks following axotomy, the number of VAChT‐ and ChAT‐immunopositive neurons had increased to almost 50% in fimbria‐fornix‐lesioned animals, indicating coordinate reexpression of both cholinergic markers in recovered neurons. There was no recovery of either VAChT or ChAT immunoreactivity after the irreversible immunotoxin lesions. Thus, with use of immunological techniques, there appears to be coordinate expression of VAChT and ChAT in the septohippocampal pathway following either unilateral fimbria‐fornix or bilateral immunotoxin lesion.
Toxic Effects of Sulphite in Combination with Peroxynitrite on Neuronal Cells
Authors: Reist Marianne , Marshall Karyn‐Ann , Jenner Peter , Halliwell Barry
Published: July 2010

Abstract: Sulphite is widely used as a preservative and antioxidant in foods, beverages, and pharmaceuticals. Endogenous sulphite is generated during the normal metabolism of sulphur‐containing amino acids, and alterations in sulphur amino acid metabolism occur in some neurodegenerative diseases. In particular, sulphite oxidase deficiency produces severe mental retardation, seizures, spastic quadriparesis, dislocated lenses, and early death. Exposure of a neuronal cell line (rat mesencephalic cells) to high levels of sulphite induced a time‐dependent decrease in viability. Peroxynitrite was also toxic to this cell line, and sulphite affected the toxicity of ONOO−. Sulphite concentrations of ≤0.5 mM markedly potentiated cell damage induced by 200 µM ONOO−. We propose that sulphite can act as a neurotoxic agent, especially in combination with peroxynitrite. Sulphite radicals may be involved in the neurotoxic effect.
Nicotine Prevents Experimental Parkinsonism in Rodents and Induces Striatal Increase of Neurotrophic Factors
Authors: Maggio Roberto , Riva Marco , Vaglini Francesca , Fornai Francesco , Molteni Raffaella , Armogida Marianna , Racagni Giorgio , Corsini Giovanni U.
Published: July 2010

Abstract: The repeated finding of an apparent protective effect of cigarette smoking on the risk of Parkinson's disease is one of the few consistent results in the epidemiology of this disorder. Among the numerous substances that originate from tobacco smoke, nicotine is by far the most widely studied. Nicotine is a natural alkaloid that has considerable stimulatory effects on the CNS. Its effects on the CNS are mediated by the activation of neuronal heteromeric acetylcholine‐gated ion channel receptors (nAChRs, also termed nicotinic acetylcholine receptors). In the present study, we describe the neuroprotective effects of (−)‐nicotine in two animal models of parkinsonism: diethyldithiocarbamate‐induced enhancement of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine toxicity in mice and methamphetamine‐induced neurotoxicity in rats and mice. The neuroprotective effect of (−)‐nicotine was very similar to that of the noncompetitive NMDA receptor antagonist (+)‐MK‐801. In parallel experiments, we found that (−)‐nicotine induces the basic fibroblast growth factor‐2 (FGF‐2) and the brain‐derived neurotrophic factor in rat striatum. The effect of (−)‐nicotine on the induction of FGF‐2 was prevented by the nAChR antagonist mecamylamine. We also found that (+)‐MK‐801 was able to induce FGF‐2 in the striatum. As trophic factors have been reported to be neuroprotective for dopaminergic cells, our data suggest that the increase in neurotrophic factors is a possible mechanism by which (−)‐nicotine protects from experimental parkinsonisms.
Effects of Aluminum and Calcium on Acetyl‐CoA Metabolism in Rat Brain Mitochondria
Authors: Szutowicz Andrzej , Bielarczyk Hanna , Kisielevski Yuri , Jankowska Agnieszka , Madziar Beata , Tomaszewicz Maria
Published: July 2010

Abstract: Al complexes are known to accumulate in extra‐ and intracellular compartments of the brain in the course of different encephalopathies. In this study possible effects of Al accumulation in the cytoplasmic compartment on mitochondrial metabolism were investigated. Al, like Ca, inhibited pyruvate utilization as well as citrate and oxoglutarate accumulation by whole brain mitochondria. Potencies of Ca2+total effects were 10–20 times stronger than those of Al. Al decreased mitochondrial acetyl‐CoA content in a concentration‐dependent manner, along with an equivalent rise of free CoA level, whereas Ca caused loss of both intermediates from mitochondria. In the absence of Pi in the medium, Ca had no effect on mitochondrial metabolism, whereas Al lost its ability to suppress pyruvate utilization and acetyl‐CoA content in Ca‐free conditions. Verapamil potentiated, whereas ruthenium red reversed, Ca‐evoked suppression of mitochondrial metabolism. On the other hand, in Ca‐supplemented medium, Al partially overcame the inhibitory influence of verapamil. Accordingly, verapamil increased mitochondrial Ca levels much more strongly than Al. However, Al partially reversed the verapamil‐evoked rise of Ca2+total level. These data indicate that Al accumulated in cytoplasm in the form of the Al(PO4)OH− complex may inhibit mitochondrial functions by an increase of intramitochondrial [Ca2+]total resulting from the Al‐evoked rise of cytoplasmic [Ca2+]free, as well as from inhibitory interference with the verapamil binding site on the Na+/Ca2+ antiporter.
Expression of NMDAR1, GluR1, GluR7, and KA1 Glutamate Receptor mRNAs Is Decreased in Frontal Cortex of “Neuroleptic‐Free” Schizophrenics: Evidence on Reversible Up‐Regulation by Typical Neuroleptics
Authors: Sokolov Boris P.
Published: July 2010

Abstract: Schizophrenics exhibit abnormalities in many memory‐associated functions mediated by the frontal cortex. Glutamate receptors play key roles in learning and memory. Hence, abnormalities in glutamate receptors within the frontal cortex may be associated with schizophrenia. In addition, emerging evidence indicates that glutamate receptors may be involved in the actions of antipsychotic drugs. To test these hypotheses, we measured mRNAs encoding the NMDAR1, GluR1, GluR7, and KA1 subunits of glutamate receptor in the left superior frontal gyrus from 21 elderly schizophrenics with varying histories of antipsychotic drug treatment and nine normal drug‐free elderly controls. There were significant negative correlations between NMDAR1, GluR1, GluR7, and KA1 mRNA levels and time without neuroleptic medication before death in schizophrenics, indicating that levels of the glutamate receptor mRNAs decline rapidly after drug withdrawal. Further analysis revealed that in “neuroleptic‐free” (>6 months) schizophrenics, levels of NMDAR1, GluR1, GluR7, and KA1 mRNAs were significantly lower than in controls. By contrast, in schizophrenics who were receiving neuroleptics until death, levels of NMDAR1, GluR1, GluR7, and KA1 mRNAs did not differ significantly from controls. These findings indicate that decreased levels of NMDAR1, GluR1, GluR7, and KA1 mRNAs may be present in the frontal cortex of some schizophrenics and that typical neuroleptics may reversibly increase levels of these mRNAs.
New Phosphorylation Sites Identified in Hyperphosphorylated Tau (Paired Helical Filament‐Tau) from Alzheimer's Disease Brain Using Nanoelectrospray Mass Spectrometry
Authors: Hanger Diane P. , Betts Joanna C. , Loviny Thérèse L. F. , Blackstock Walter P. , Anderton Brian H.
Published: July 2010

Abstract: Paired helical filaments (PHFs) are the structural constituents of neurofibrillary tangles in Alzheimer's disease and are composed of hyperphosphorylated forms of the microtubule‐associated protein tau (PHF‐tau). Pathological hyperphosphorylation of tau is believed to be an important contributor to the destabilisation of microtubules and their subsequent disappearance from tangle‐bearing neurons in Alzheimer's disease, making elucidation of the mechanisms that regulate tau phosphorylation an important research goal. Thus, it is essential to identify, preferably by direct sequencing, all of the sites in PHF‐tau that are phosphorylated, a task that is incomplete because of the difficulty to date of purifying insoluble PHF‐tau to homogeneity and in sufficient quantities for structural analysis. Here we describe the solubilisation of PHF‐tau followed by its purification by Mono Q chromatography and reversed‐phase HPLC. Phosphopeptides from proteolytically digested PHF‐tau were sequenced by nanoelectrospray mass spectrometry. We identified 22 phosphorylation sites in PHF‐tau, including five sites not previously identified. The combination of our new data with previous reports shows that PHF‐tau can be phosphorylated on at least 25 different sites.
Excitatory Amino Acid Synthesis in Hypoxic Brain Slices: Does Alanine Act as a Substrate for Glutamate Production in Hypoxia?
Authors: Griffin J. L. , Rae C. , Dixon R. M. , Radda G. K. , Matthews P. M.
Published: July 2010

Abstract: Excitatory amino acids are an important cause of cell death in the hypoxic and ischaemic brain. Neuronal glutamate stores are depleted rapidly in hypoxia, but alanine production rises under such conditions and has been suggested to be a potential precursor of glutamate. To test this hypothesis, we have investigated amino acid metabolism using 13C NMR with superfused guinea pig cortical slices subjected to varying degrees of hypoxia. During severe hypoxia, brain slices metabolising 5 mM [2‐13C]pyruvate exported [2‐13C]alanine into the superfusion fluid. The metabolic fate of alanine during normoxia and hypoxia was tested by superfusion of brain slices with 10 mM glucose and 2 mM [2‐13C, 15N]alanine. Metabolism of exogenous alanine leads to the release of aspartate into the superfusion fluid. The pattern of labelling of aspartate indicated that it was synthesised via the glial‐specific enzyme pyruvate carboxylase. 13C‐labelled glutamate was produced with both normoxia and hypoxia, but concentrations were 30‐fold lower than for labelled aspartate. Thus, although substantial amounts of glutamate are not synthesised from alanine in hypoxia, there is significant production of aspartate, which also may have deleterious effects as an excitatory amino acid.
Characterization of (2S,2′R,3′R)‐2‐(2′,3′‐[3H]‐Dicarboxycyclopropyl)glycine Binding in Rat Brain
Authors: Mutel Vincent , Adam Geo , Chaboz Sylvie , Kemp John A. , Klingelschmidt Agnés , Messer Jürg , Wichmann Jürgen , Woltering Thomas , Richards John Grayson
Published: July 2010

Abstract: [(2S,2′R,3′R)‐2‐(2′,3′‐[3H]Dicarboxycyclopropyl)glycine ([3H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a KD value of 180 ± 33 nM and a Bmax of 780 ± 70 fmol/mg of protein. The nonspecific binding, measured using 100 µM LY354740, was <30%. NMDA, AMPA, kainate, l(−)‐threo‐3‐hydroxyaspartic acid, and (S)‐3,5‐dihydroxyphenylglycine were all inactive in [3H]DCG IV binding up to 1 mM. However, several compounds inhibited [3H]DCG IV binding in a concentration‐dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1′S,2′S)‐2‐(2‐carboxycyclopropyl)glycine > (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid > (2S,1′S,2′S)‐2‐methyl‐2‐(2‐carboxycyclopropyl)glycine > l‐glutamate = ibotenate > quisqualate > (RS)‐α‐methyl‐4‐phosphonophenylglycine = l(+)‐2‐amino‐3‐phosphonopropionic acid > (S)‐α‐methyl‐4‐carboxyphenylglycine > (2S)‐α‐ethylglutamic acid > l(+)‐2‐amino‐4‐phosphonobutyric acid. N‐Acetyl‐l‐aspartyl‐l‐glutamic acid inhibited the binding in a biphasic manner with an IC50 of 0.2 µM for the high‐affinity component. The binding was also affected by GTPγS, reducing agents, and CdCl2. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre‐ and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPγS show that [3H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.
Excitotoxin‐Induced Neuronal Death Is Associated with Response of a Unique Intracellular Aspartic Proteinase, Cathepsin E
Authors: Tominaga Kazuyoshi , Nakanishi Hiroshi , Yasuda Yoshiyuki , Yamamoto Kenji
Published: July 2010

Abstract: Excitotoxicity produced by excessive stimulation of glutamate receptors is known to lead to neuronal lesion and death. Here, we demonstrate that quantitative and qualitative changes in cathepsin E (CE) gene products are associated with execution of the excitotoxic neuronal death. Intracerebroventricular injection of kainate (KA) resulted in marked elevation of both mRNA and protein levels of CE in the rat hippocampal CA3 region, where the enzyme was mainly found in vulnerable neurons and activated microglia. Northern blot analysis showed that the size of the transcript for CE was identical with that normally expressed in rat spleen. Immunoblot analysis, however, revealed the predominant occurrence of the highly modified CE species, besides the mature CE. This polypeptide was distinct from the mature CE in molecular masses (106 vs. 82 kDa) and pl (6.4–7.3 vs. 5.0–5.5) and showed resistance to conversion into the enzymatically active form by acid treatment. Consistent with these in vivo results, administration of glutamate to primary cultured rat hippocampal neurons resulted in a marked expression of this novel CE species. These data indicate that excessive stimulation of glutamate receptors causes induction of the CE gene response followed by persistent expression of CE in the novel form, besides its mature form, predominantly in the hippocampal neurons.
Cross‐Linking Sites of the Human Tau Protein, Probed by Reactions with Human Transglutaminase
Authors: Prasanna Murthy S. N. , Wilson James H. , Lukas Thomas J. , Kuret Jeff , Lorand Laszlo
Published: July 2010

Abstract: A portion of the neurofibrillary tangles of Alzheimer's disease has the characteristics of cross‐linked protein. Because the principal component of these lesions is the microtubule‐associated protein tau, and because a major source of cross‐linking activity within neurons is supplied by tissue transglutaminase (TGase), it has been postulated that isopeptide bond formation is a major posttranslational modification leading to the formation of insoluble neurofibrillary tangles. Here we have mapped the sites on two isoforms of human tau protein (τ23 and τ40) capable of participating in human TGase‐mediated isopeptide bond formation. Using dansyl‐labeled fluorescent probes, it was shown that eight Gln residues can function as amine acceptor residues, with two major sites being Gln351 and Gln424. In addition, 10 Lys residues were identified as amine donors, most of which are clustered adjacent to the microtubule‐binding repeats of tau in regions known to be solvent accessible in filamentous tau. The distribution of amine donors correlated closely with that of Arg residues, suggesting a link between neighboring positive charge and the TGase selectivity for donor sites in the protein substrate. Apart from revealing the sites that can be cross‐linked during the TGase‐catalyzed assembly of tau filaments, the results suggest a topography for the tau monomers so assembled.
Increased Vulnerability to 3‐Nitropropionic Acid in an Animal Model of Huntington's Disease
Authors: Bogdanov Mikhail B. , Ferrante Robert J. , Kuemmerle Stefan , Klivenyi Peter , Beal M. Flint
Published: July 2010

Abstract: There is substantial evidence for both metabolic dysfunction and oxidative damage in Huntington's disease (HD). In the present study, we used in vivo microdialysis to measure the conversion of 4‐hydroxybenzoic acid to 3,4‐dihydroxybenzoic acid (3,4‐DHBA) as a measure of hydroxyl radical production in a transgenic mouse model of HD, as well as in littermate controls. The conversion of 4‐hydroxybenzoic acid to 3,4‐DHBA was unchanged in the striatum of transgenic HD mice at baseline. Following administration of the mitochondrial toxin 3‐nitropropionic acid (3‐NP), there were significant increases in 3,4‐DHBA generation in both control and transgenic HD mice, and the increases in the transgenic HD mice were significantly greater than those in controls. Furthermore, administration of 3‐NP produced significantly larger striatal lesions in transgenic HD mice than in littermate controls. The present results show increased sensitivity to the mitochondrial toxin 3‐NP in transgenic HD mice, which suggests metabolic dysfunction in this mouse model of HD.
Up‐Regulation of Base Excision Repair Activity for 8‐Hydroxy‐2'‐Deoxyguanosine in the Mouse Brain After Forebrain Ischemia‐Reperfusion
Authors: Lin Lie‐Huey , Cao Shutong , Yu Ling , Cui Jiankun , Hamilton Winifred J. , Liu Philip K.
Published: July 2010

The repair enzyme 8‐oxoguanine glycosylase/apyrimidinic/apurinic lyase (OGG) removes 8‐hydroxy‐2′‐deoxyguanosine (oh8dG) in human cells. Our goal was to examine oh8dG‐removing activity in the cell nuclei of male C57BL/6 mouse brains treated with either forebrain ischemia‐reperfusion (FbIR) or sham operations. We found that the OGG activity in nuclear extracts, under the condition in which other nucleases did not destroy the oligodeoxynucleotide duplex, excised oh8dG with the greatest efficiency on the oligodeoxynucleotide duplex containing oh8dG/dC and with less efficiency on the heteroduplex containing oh8dG/dT, oh8dG/dG, or oh8dG/dA. This specificity was the same as for the recombinant type 1 OGG (OGG1) of humans. We observed that the OGG1 peptide and its activity in the mouse brain were significantly increased after 90 min of ischemia and 20‐30 min of reperfusion. The increase in the protein level and in the activity of brain OGG1 correlated positively with the elevation of FbIR‐induced DNA lesions in an indicator gene (the c‐fos gene) of the brain. The data suggest a possibility that the OGG1 protein may excise oh8dG in the mouse brain and that the activity of OGG1 may have a functional role in reducing oxidative gene damage in the brain after FbIR.
Elevated N1‐Acetylspermidine Levels in Gerbil and Rat Brains After CNS Injury
Authors: Rao A. Muralikrishna , Hatcher James F. , Dogan Aclan , Dempsey Robert J.
Published: July 2010

The polyamine system is very sensitive to different pathological states of the brain and is perturbed after CNS injury. The main modifications are significant increases in ornithine decarboxylase activity and an increase in tissue putrescine levels. Previously we have shown that the specific polyamine oxidase (PAO) inhibitor N1,N4‐bis(2,3‐butadienyl)‐1,4‐butanediamine (MDL 72527) reduced the tissue putrescine levels, edema, and infarct volume after transient focal cerebral ischemia in spontaneously hypertensive rats and traumatic brain injury of Sprague‐Dawley rats. In the present study, N1‐acetylspermidine accumulation was greater in injured brain regions compared with sham or contralateral regions following inhibition of PAO by MDL 72527. This indicates spermidine/spermine‐N1‐acetyltransferase (SSAT) activation after CNS injury. The observed increase in N1‐acetylspermidine levels at 1 day after CNS trauma paralleled the decrease in putrescine levels after treatment with MDL 72527. This suggests that the increased putrescine formation at 1 day after CNS injury is mediated by the SSAT/PAO pathway, consistent with increased SSAT mRNA after transient ischemia.
Substrate‐Bound β‐Amyloid Peptides Inhibit Cell Adhesion and Neurite Outgrowth in Primary Neuronal Cultures
Authors: Postuma Ronald B. , He Weilan , Nunan Janelle , Beyreuther Konrad , Masters Colin L. , Barrow Colin J. , Small David H.
Published: July 2010

Accumulation of theβ‐amyloid protein (Aβ) in the brain is an important step in the pathogenesis of Alzheimer's disease. However, the mechanism of Aβ toxicity remains unclear. Aβ can bind to the extracellular matrix, a structure that regulates adhesive events such as neurite outgrowth and synaptogenesis. The binding of Aβ to the extracellular matrix suggests that Aβ may disrupt cell‐substrate interactions. Therefore, the effect of substrate‐bound Aβ on the growth of isolated chick sympathetic and mouse cortical neurons was examined. Aβ1‐40 and Aβ1‐42 had dose‐dependent effects on cell morphology. When tissue culture plates were coated with 0.1‐10 ng/well Aβ, neurite outgrowth increased. Higher amounts of Aβ peptides (≥μg/well) inhibited outgrowth. The inhibitory effect was related to aggregation of the peptide, as preincubation of Aβ1‐40 for 24 h at 37 °C (a process known to increase amyloid fibril formation) was necessary for inhibition of neurite outgrowth. Aβ29‐42, but not Aβ1‐28, also inhibited neurite outgrowth at high concentrations, demonstrating that the inhibitory domain is located within the hydrophobic C‐terminal region. Aβ1‐40, Aβ1‐42, and Aβ29‐42 also inhibited cell‐substrate adhesion, indicating that the effect on neurite outgrowth may have been due to inhibition of cell adhesion. The results suggest that accumulation of Aβ may disrupt cell‐adhesion mechanisms in vivo.