Journal of Neurochemistry

Prolonged activation of ERK triggers glutamate-induced apoptosis of astrocytes: neuroprotective effect of FK506.

Publication Date: 2010 Feb 25 PMID: 20202085
Authors: Szydlowska, K. - Gozdz, A. - Dabrowski, M. - Zawadzka, M. - Kaminska, B.
Journal: J Neurochem

ABSTRACT Although, astrocytes are more resistant than neurons to ischemic injury, astrocyte death has been demonstrated in animal models of brain ischemia. Astrocytes death after ischemia/reperfusion may strongly affect neuronal survival due to absence of their trophic and metabolic support to neurons, and astrocytic glutamate uptake. Early signals involved in astrocytes death are poorly understood. We demonstrated enhanced and mostly cytoplasmic activation of Extracellular Signal-Regulated kinases 1 and 2 (ERK1/2) during glutamate-induced apoptosis of cultured astrocytes. Treatment with UO126, inhibitor of MEK1, TBOA, glutamate transporter inhibitor, and FK506, a cytoprotective drug FK506 prevented ERK activation and glutamate-induced apoptosis. Overexpression of ERK phosphatases DUSP5 and DUSP6 reduced apoptosis in transfected astrocytes. Prolonged ERK1/2 activation was observed in ischemic brain: in the nucleus and cytoplasm of astrocytes in the cerebral cortex, and exclusively in the cytoplasm of astrocytes in the striatum. Global gene expression profiling in the cortex revealed that FK506 blocks MCAo-induced expression of numerous genes associated with ERK signaling pathway and apoptosis. The results demonstrate a pro-apoptotic role of sustained activation of ERK1/2 signaling in glutamate-induced death of astrocytes and the ability of FK506 to block both ERK activation and astrocytic cell death in vitro and in ischemic brains.

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Quantal release of acetylcholine in mice with reduced levels of the vesicular acetylcholine transporter.

Publication Date: 2010 Feb 25 PMID: 20202084
Authors: de Freitas Lima, R. - Prado, V. F. - Prado, M. A. - Kushmerick, C.
Journal: J Neurochem

Abstract Mammalian motor nerve terminals contain hundreds of thousands of synaptic vesicles, but only a fraction of these vesicles is immediately available for release, the remainder forming a reserve pool. The supply of vesicles is replenished through endocytosis, and newly formed vesicles are refilled with acetylcholine through a process that depends on the vesicular acetylcholine transporter (VAChT). During expression of short-term plasticity, quantal release can be increased, but it is unknown whether this reflects enhanced recruitment of vesicles from the reserve pool or rapid recycling. We examined spontaneous and evoked release of acetylcholine at endplates from genetically modified VAChT KD(HOM) mice that express approximately 30% of the normal level of VAChT in order to determine steps rate-limited by synaptic vesicle filling. Quantal content and quantal size were reduced in VAChT KD(HOM) mice compared to wild-type controls. Although high-frequency stimulation did not reduce quantal size further, the post-tetanic increase in EPP amplitude or MEPP frequency was significantly smaller in VAChT KD(HOM) mice. This was the case even when tetanic depression was eliminated using an extracellular solution containing reduced Ca(2+) and raised Mg(2+). These results reveal the dependence of short-term plasticity on the level of VAChT expression and efficient synaptic vesicle filling.

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DJ-1 forms complexes with mutant SOD1 and ameliorates its toxicity.

Publication Date: 2010 Feb 25 PMID: 20202083
Authors: Yamashita, S. - Mori, A. - Kimura, E. - Mita, S. - Maeda, Y. - Hirano, T. - Uchino, M.
Journal: J Neurochem

Abstract Mutations in Cu/Zn superoxide dismutase (SOD1) gene cause familial amyotrophic lateral sclerosis (ALS), which could be attributed to the toxic properties of the misfolded protein, oxidative stress, and mitochondrial dysfunction. DJ-1-a causative agent of familial Parkinson's disease PARK7-is responsible for inducing antioxidative reaction. Here, we showed the upregulation of DJ-1 protein levels in mutant SOD1 transgenic mice through the lifespan were observed in the motor neurons. We demonstrated biochemically DJ-1 formed complexes with mutant SOD1 in the cell lysates. Furthermore, DJ-1 overexpression resulted in increased cell viability and reduced cell toxicity in mutant SOD1-transfected neuronal cells, because of improvement in apoptotic pathway and reduction in oxidative stress levels. We also evaluated DJ-1 levels in cerebrospinal fluid (CSF) collected from sporadic ALS patients and controls subjects. The CSF DJ-1 levels were significantly higher in patients with sporadic ALS than in control subjects. These results show that DJ-1 may be associated with sporadic and familial ALS pathogenesis. Therefore, insight into the effects of DJ-1 on mutant SOD1-mediated toxicity may provide a therapeutic advance for the treatment of motor neuron degeneration in ALS.

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The hematopoietic factor Granulocyte-colony stimulating factor (G-CSF) improves outcome in experimental spinal cord injury.

Publication Date: 2010 Feb 25 PMID: 20202082
Authors: Pitzer, C. - Klussmann, S. - Kruger, C. - Letellier, E. - Plaas, C. - Dittgen, T. - Kirsch, F. - Stieltjes, B. - Weber, D. - Laage, R. - Martin-Villalba, A. - Schneider, A.
Journal: J Neurochem

Abstract Granulocyte-colony stimulating factor (G-CSF) is a potent hematopoietic factor that drives differentiation of neutrophilic granulocytes. We have recently shown that G-CSF also acts as a neuronal growth factor, protects neurons in vitro and in vivo, and has regenerative potential in various neurological disease models. Spinal cord injury following trauma or secondary to skeletal instability is a terrible condition with no effective therapies available at present. Here, we show that the G-CSF receptor is upregulated upon experimental spinal cord injury and that G-CSF improves functional outcome in a partial dissection model of spinal cord injury. G-CSF significantly decreases apoptosis in an experimental partial spinal transsection model in the mouse and increases expression of the anti-apoptotic G-CSF target gene Bcl-X(L). In vitro, G-CSF enhances neurite outgrowth and branching capacity of hippocampal neurons. In vivo, G-CSF treatment results in improved functional connectivity of the injured spinal cord as measured by Mn(2+)-enhanced MRI. G-CSF also increased length of the dorsal corticospinal tract and density of serotonergic fibers cranial to the lesion center. Mice treated systemically with G-CSF as well as transgenic mice overexpressing G-CSF in the CNS exhibit a strong improvement in functional outcome as measured by the BBB score and gridwalk analysis. We show that G-CSF improves outcome after experimental spinal cord injury by counteracting apoptosis, and enhancing connectivity in the injured spinal cord. We conclude that G-CSF constitutes a promising and feasible new therapy option for SCI.

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Hemopressins and other hemoglobin-derived peptides in mouse brain: Comparison between brain, blood, and heart peptidome and regulation in Cpe(fat/fat) mice.

Publication Date: 2010 Feb 24 PMID: 20202081
Authors: Gelman, J. S. - Sironi, J. - Castro, L. M. - Ferro, E. S. - Fricker, L. D.
Journal: J Neurochem

ABSTRACT Many hemoglobin-derived peptides are present in mouse brain, and several of these have bioactive properties including the hemopressins, a related series of peptides that bind to cannabinoid CB1 receptors. Although hemoglobin is a major component of red blood cells, it is also present in neurons and glia. To examine whether the hemoglobin-derived peptides in brain are similar to those present in blood and heart, we used a peptidomics approach involving mass spectrometry. Many hemoglobin-derived peptides are found only in brain and not in blood, whereas all hemoglobin-derived peptides found in heart were also seen in blood. Thus, it is likely that the majority of the hemoglobin-derived peptides detected in brain are produced from brain hemoglobin and not erythrocytes. We also examined if the hemopressins and other major hemoglobin-derived peptides were regulated in the Cpe(fat/fat) mouse; previously these mice were reported to have elevated levels of several hemoglobin-derived peptides. Many, but not all of the hemoglobin-derived peptides were elevated in several brain regions of the Cpe(fat/fat) mouse. Taken together, these findings suggest that the post-translational processing of alpha and beta hemoglobin into the hemopressins, as well as other peptides, is upregulated in some but not all Cpe(fat/fat) mouse brain regions.

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Nicotine self-administration differentially modulates glutamate and GABA transmission in hypothalamic paraventricular nucleus to enhance the hypothalamic-pituitary-adrenal response to stress.

Publication Date: 2010 Feb 24 PMID: 20202080
Authors: Yu, G. - Chen, H. - Wu, X. - Matta, S. G. - Sharp, B. M.
Journal: J Neurochem

Abstract The Mechanisms by which chronic nicotine self-administration augments hypothalamo-pituitary-adrenal (HPA) responses to stress are only partially understood. Nicotine self-administration alters neuropeptide expression in corticotropin-releasing factor (CRF) neurons within paraventricular nucleus (PVN) and increases PVN responsiveness to norepinephrine during mild footshock stress. Glutamate and GABA also modulate CRF neurons, but their roles in enhanced HPA responsiveness to footshock during chronic self-administration are unknown. We show that nicotine self-administration augmented footshock-induced PVN glutamate release, but further decreased GABA release. In these rats, intra-PVN kynurenic acid, a glutamate receptor antagonist, blocked enhanced adrenocorticotropic hormone and corticosterone responses to footshock. In contrast, peri-PVN kynurenic acid, which decreases activity of GABA afferents to PVN, enhanced footshock-induced corticosterone secretion only in control rats self-administering saline. Additionally, in rats self-administering nicotine, footshock-induced elevation of corticosterone was significantly less than in controls after intra-PVN saclofen (GABA-B receptor antagonist). Therefore, the exaggerated reduction in GABA release by footshock during nicotine self-administration disinhibits CRF neurons. This disinhibition combined with enhanced glutamate input provides a new mechanism for HPA sensitization to stress by chronic nicotine self-administration. This mechanism, which does not preserve homeostatic plasticity, supports the concept that smoking functions as a chronic stressor that sensitizes the HPA to stress.

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Eph receptor tyrosine kinases regulate astrocyte cytoskeletal rearrangement and focal adhesion formation.

Publication Date: 2010 Feb 24 PMID: 20202079
Authors: Puschmann, T. B. - Turnley, A. M.
Journal: J Neurochem

Abstract EphA4 null mice have impaired astrocytic gliosis following spinal cord injury. This may be due to altered cytoskeletal regulation and is examined herein using cultured astrocytes from wildtype and EphA4 null mice. Under basal conditions EphA4 null astrocytes appeared relatively normal but following stimuli resulting in cytoskeletal rearrangement, EphA4 null cells responded more slowly. When F-actin stress fibers were collapsed using the Rho kinase inhibitor HA1077, fewer EphA4 null cells showed stress fiber collapse in response to HA1077 and recovered stress fibers more slowly following HA1077 removal. EphA4 null astrocytes were less adherent and had smaller focal adhesions, while activation of Eph receptors with ephrin-A5-Fc increased the numbers of focal adhesions in both wildtype and knockout astrocytes following serum starvation. Using scratch wound assays, EphA4 null astrocytes invading the scratch showed impaired GFAP expression, particularly in proliferative cells. Astrocytes did not express Ephexin, a major Eph-interacting Rho guanine exchange factor. but they expressed Vav proteins, with lower levels of phospho-Vav in EphA4 null compared to wildtype astrocytes. This may contribute to the slower cytoskeletal responses generally observed in the EphA4 null astrocytes. Eph receptor signaling therefore regulates astrocyte reactivity through modulation of cytoskeletal responses.

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Modulation of bone morphogenic protein signalling alters numbers of astrocytes and oligodendroglia in the subventricular zone during cuprizone-induced demyelination.

Publication Date: 2010 Feb 27 PMID: 20193041
Authors: Cate, H. S. - Sabo, J. K. - Merlo, D. - Kemper, D. - Aumann, T. D. - Robinson, J. - Merson, T. D. - Emery, B. - Perreau, V. M. - Kilpatrick, T. J.
Journal: J Neurochem

The adult subventricular zone (SVZ) is a potential source of precursor cells to replace neural cells lost during demyelination. To better understand the molecular events that regulate neural precursor cell responsiveness in this context we undertook a microarray and quantitative PCR based analysis of genes expressed within the SVZ during cuprizone-induced demyelination. We identified an upregulation of the genes encoding Bone Morphogenic Protein 4 (BMP4) and its receptors. Immunohistochemistry confirmed an increase in BMP4 protein levels and also showed an increase in phosphorylated SMAD 1/5/8, a key component of BMP4 signalling, during demyelination. In vitro analysis revealed that neural precursor cells isolated from demyelinated animals, as well as those treated with BMP4, produce more astrocytes. Similarly, there were increased numbers of astrocytes in vivo within the SVZ during demyelination. Intraventricular infusion of Noggin, an endogenous antagonist of BMP4, during cuprizone-induced demyelination reduced pSMAD1/5/8, decreased astrocyte numbers and increased oligodendrocyte numbers in the SVZ. Our results suggest that lineage commitment of SVZ neural precursor cells is altered during demyelination and that BMP signalling plays a role in this process.

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Increased expression of cholesterol 24S-hydroxylase results in disruption of glial glutamate transporter EAAT2 association with lipid rafts: a potential role in Alzheimer's disease.

Publication Date: 2010 Feb 27 PMID: 20193040
Authors: Tian, G. - Kong, Q. - Lai, L. - Ray-Chaudhury, A. - Liang Glenn Lin, C.
Journal: J Neurochem

The glial glutamate transporter EAAT2 is the major mediator of glutamate clearance that terminates glutamate-mediated neurotransmission. Loss of EAAT2 and associated glutamate uptake function has been reported in the brains of patients with Alzheimer's disease (AD). We previously reported that EAAT2 is associated with lipid raft microdomains of the plasma membrane. In the present study, we demonstrated that association of EAAT2 with lipid rafts is disrupted in AD brains. This abnormality is not a consequence of neuron degeneration, oxidative stress, or amyloid beta toxicity. In AD brains, cholesterol 24S-hydroxylase (CYP46), a key enzyme in maintenance of cholesterol homeostasis in the brain, is markedly increased in astrocytes but decreased in neurons. We demonstrated that increased expression of CYP46 in primary astrocytes results in a reduction of membrane cholesterol levels and leads to the dissociation of EAAT2 from lipid rafts and the loss of EAAT2 and associated glutamate uptake function. These results suggest that a disturbance of cholesterol metabolism may contribute to loss of EAAT2 in AD.

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Human TrkB gene: novel alternative transcripts, protein isoforms and expression pattern in the prefrontal cerebral cortex during postnatal development.

Publication Date: 2010 Feb 27 PMID: 20193039
Authors: Luberg, K. - Wong, J. - Weickert, C. S. - Timmusk, T.
Journal: J Neurochem

Brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) high-affinity receptor tropomyosine related kinase B (TrkB) is required for the differentiation and maintenance of specific neuron populations. Misregulation of TrkB has been reported in many human diseases, including cancer, obesity and neurological and psychiatric disorders. Alternative splicing that generates receptor isoforms with different functional properties also regulates TrkB function. Here we describe numerous novel isoforms of TrkB proteins, including isoforms generated by alternative splicing of cassette exons in the regions encoding both the extracellular and intracellular domain and also N-terminally truncated isoforms encoded by novel 5' exon-containing transcripts. We also characterize the intracellular localization and phosphorylation potential of novel TrkB isoforms and find that these proteins have unique properties. Additionally, we describe the expression profiles of all the known human TrkB transcripts in adult tissues and also during postnatal development in the human prefrontal cortex. We show that transcripts encoding the full-length TrkB receptor and the C-terminally truncated TrkB-T1 have different expression profiles as compared to the proteins they encode. Identification of 36 potential TrkB protein isoforms suggests high complexity in the synthesis, regulation and function of this important neurotrophin receptor emphasizing the need for further study of these novel TrkB variants.

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Drosophila models of human tauopathies indicate that Tau protein toxicity in vivo is mediated by soluble cytosolic phosphorylated forms of the protein.

Publication Date: 2010 Feb 27 PMID: 20193038
Authors: Feuillette, S. - Miguel, L. - Frebourg, T. - Campion, D. - Lecourtois, M.
Journal: J Neurochem

Tau is a neuronal microtubule-associated protein involved in microtubules assembly and stabilization. Tauopathies, including Alzheimer's disease and fronto-temporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), are a group of neurodegenerative disorders characterized by the presence of intraneuronal filamentous inclusions of abnormally and hyperphosphorylated Tau. Currently, the molecular mechanisms underlying Tau-mediated cellular toxicity remain elusive. To address the determinants of Tau neurotoxicity, we used Drosophila models of human tauopathies to study the microtubule-binding properties of human Tau proteins in vivo. We showed that, in contrast to endogenous Drosophila Tau, human Tau proteins bind very poorly to microtubules in Drosophila, and are mostly recovered as soluble cytosolic hyperphosphorylated species. This weak binding of human Tau to microtubules is neither due to microtubules saturation nor competition with endogenous Drosophila Tau, but clearly depends on its phosphorylation degree. We also reported that accumulation of cytosolic hyperphosphorylated forms of human Tau proteins correlates with human Tau-mediated neurodegeneration in flies, supporting the key role of soluble cytosolic hyperphosphorylated Tau proteins as toxic species in vivo.

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Evidence for two conductive pathways in P2X(7) receptor: differences in modulation and selectivity.

Publication Date: 2010 Feb 17 PMID: 20180885
Authors: Alloisio, S. - Di Garbo, A. - Barbieri, R. - Bozzo, L. - Ferroni, S. - Nobile, M.
Journal: J Neurochem

ABSTRACT The P2X(7) receptor (P2X(7)R) is an ATP-gated cation channel whose biophysical properties remain to be unravelled unequivocally. Its activity is modulated by divalent cations and organic messengers such as arachidonic acid (AA). In this study we analysed the differential modulation of magnesium (Mg(2+)) and AA on P2X(7)R by measuring whole-cell currents and intracellular Ca(2+) ([Ca(2+)](i)) and Na(+) ([Na(+)](i)) dynamics in HEK293 cells stably expressing full-length P2X(7)R and in cells endowed with the P2X(7)R variant lacking the entire C-terminus tail (trP2X(7)R), which is thought to control the pore activation. AA induced a robust potentiation of the P2X(7)R- and trP2X(7)R-mediated [Ca2+]i rise but did not affect the ionic currents in both conditions. Extracellular Mg(2+) reduced the P2X7R- and trP2X(7)R-mediated [Ca(2+)](i) rise in a dose-dependent manner through a competitive mechanism. The modulation of the magnitude of the P2X(7)R-mediated ionic current and [Na(+)](i) rise were strongly dependent on Mg(2+) concentration but occurred in a non-competitive manner. By contrast in cells expressing the trP2X(7)R the small ionic currents and [Na(+)](i) signals were totally insensitive to Mg(2+). Collectively, these results support the tenet of a functional structure of P2X(7)R possessing at least two distinct conductive pathways one for Ca(2+) and another for monovalent ions, with the latter which depends on the presence of the receptor C-terminus.

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Corrigendum.

Publication Date: 2010 Feb 17 PMID: 20180884
Authors:
Journal: J Neurochem

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Nuclear factor-kappaB activation regulates Cyclooxygenase-2 induction in human astrocytes in response to CXCL12: Role in neuronal toxicity.

Publication Date: 2010 Feb 17 PMID: 20180883
Authors: Alvarez, S. - Blanco, A. - Fresno, M. - Munoz-Fernandez, M. A.
Journal: J Neurochem

Abstract Neurodegenerative and neuroinflammatory disorders are commonly associated with local chemokine release. In other way, emerging data indicate that the prostaglandin E2 (PGE(2)), one of the major prostaglandins produced in the brain, play a central role in several pathological diseases. Here, we have investigated the relationship between CXCL12, cyclooxygenase-2 (COX-2) and PGE(2) in human brain cells. CXCL12 induced COX-2 and secretion of PGE(2) in a dose-dependent manner in human astrocytes. This induction was abolished by treatment with PTx and AMD3100, confirming the role of CXCR4 signaling. The NF-kappaB involvement was confirmed by using PDTC, and with transient transfection assays. Overexpression of IkappaBalpha abrogated COX-2 induction, and CXCL12 induced p65/relA translocation. Culture supernatants from CXCL12-treated astrocytes reduced viability of neuroblastoma cells, and COX inhibitors abrogated this toxicity. Therefore, the relationship between chemokines and PGs could differentially influence the pathogenic network responsible for neurodegeneration.

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Abnormal Post-Translational and Extracellular Processing of Brevican in Plaque-Bearing Mice Overexpressing APPsw.

Publication Date: 2010 Feb 17 PMID: 20180882
Authors: Ajmo, J. M. - Bailey, L. A. - Howell, M. D. - Cortez, L. K. - Pennypacker, K. R. - Mehta, H. N. - Morgan, D. - Gordon, M. N. - Gottschall, P. E.
Journal: J Neurochem

ABSTRACT Aggregation of amyloid-beta in the forebrain of Alzheimer's disease subjects may disturb the molecular organization of the extracellular microenvironment that modulates neural and synaptic plasticity. Proteoglycans are major components of this extracellular environment. To test the hypothesis that amyloid-beta, or another amyloid precursor protein dependent mechanism modifies the accumulation and/or turnover of extracellular proteoglycans, we examined whether the expression and processing of brevican, an abundant extracellular, chondroitin sulfate-bearing proteoglycan, were altered in brains of amyloid-beta-depositing transgenic mice (APPsw) as a model of Alzheimer's disease. The molecular size of chondroitin sulfate chains attached to brevican was smaller in hippocampal tissue from APPsw mice bearing amyloid-beta deposits compared to non-transgenic mice, likely due to changes in the chondroitin sulfate chains. Also, the abundance of the major proteolytic fragment of brevican was markedly diminished in extracts from several telencephalic regions of APPsw mice compared to non-transgenic mice, yet these immunoreactive fragments appeared to accumulate adjacent to the plaque edge. These results suggest that amyloid-beta or amyloid precursor protein exert inhibitory effects on proteolytic cleavage mechanisms responsible for synthesis and turnover of proteoglycans. Since proteoglycans stabilize synaptic structure and inhibit molecular plasticity, defective brevican processing observed in amyloid-beta-bearing mice and potentially end-stage human Alzheimer's disease, may contribute to deficient neural plasticity.

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Human neuroblastoma cells with MYCN amplification are selectively resistant to oxidative stress by transcriptionally up-regulating glutamate cysteine ligase.

Publication Date: 2010 Feb 17 PMID: 20180881
Authors: de Tudela, M. V. - Delgado-Esteban, M. - Cuende, J. - Bolanos, J. P. - Almeida, A.
Journal: J Neurochem

Abstract Neuroblastoma is a sympathetic nervous system tumour whose degree of malignancy, prognosis and therapy resistance has been associated with the amplification of MYCN oncogene. However, the molecular pathway responsible for such resistance is unknown. To contribute addressing this issue, here we have compared the vulnerability of four human neuroblastoma cell lines differentially amplifying MYCN, namely SK-N-BE-2 and IMR-32 (MYCN-amplified cells) and SH-SY5Y and SK-N-SH (MCYN-non-amplified cells), to H(2)O(2)-mediated apoptotic death. We found that the high resistance of the MYCN-amplified neuroblastoma cells against oxidative damage can be accounted for by their greater expression of both the mRNA and protein of the catalytic subunit of glutamate-cysteine ligase (GCL(cat)), the rate-limiting step in glutathione (GSH) biosynthesis. Furthermore, we found that MYCN directly binds to an E-box containing GCL(cat) promoter and that over-expression of MYCN in MYCN-non-amplified cells stimulated GCL(cat) expression and provided resistance to oxidative damage; whereas knock down of MYCN in MYCN-amplified cells decreased GCL(cat) expression and sensitized them to oxidative damage. Finally, GCL(cat) knock down enhanced the vulnerability of MYCN- amplified cells to oxidative damage. These results demonstrate that regulation of GCL(cat) by MYCN accounts for the survival of neuroblastoma cells against oxidative damage, and suggest that GCL should be considered a potential therapeutic target for the treatment of MYCN- amplified neuroblastoma.

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AN UNC-49 GABA RECEPTOR SUBUNIT FROM THE PARASITIC NEMATODE Haemonchus contortus IS ASSOCIATED WITH ENHANCED GABA SENSITIVITY IN NEMATODE HETEROMERIC CHANNELS.

Publication Date: 2010 Feb 23 PMID: 20180830
Authors: Siddiqui, S. Z. - Brown, D. D. - Rao, V. T. - Forrester, S. G.
Journal: J Neurochem

We have identified two genes from the parasitic nematode Haemonchus contortus, Hco-unc-49B and Hco-unc-49C that encode two GABA-gated chloride channel subunits. Electrophysiological analysis revealed that this channel has properties similar to those of the UNC-49 channel from the free-living nematode Caenorhabditis elegans. For example, the Hco-UNC-49B subunit forms a functional homomeric channel that responds to GABA and is highly sensitive to picrotoxin. Hco-UNC-49C alone does not respond to GABA but can assemble with Hco-UNC-49B to form a heteromeric channel with a lower sensitivity to picrotoxin. However, we did find that the Hco-UNC-49B/C heteromeric channel is significantly more responsive to agonists compared to the Hco-UNC-49B homomeric channel, which is the opposite trend to what has been found previously for the C. elegans channel. To investigate the subunit requirements for high agonist sensitivity, we generated cross-assembled channels by co-expressing the H. contortus subunits with UNC-49 subunits from C. elegans (Cel-UNC-49). Co-expressing Cel-UNC-49B with Hco-UNC-49C produced a heteromeric channel with a reduced sensitivity to GABA compared to that of the Cel-UNC-49B homomeric channel. In contrast, co-expressing Hco-UNC-49B with Cel-UNC-49C produced a heteromeric channel that, like the Hco-UNC-49B/C heteromeric channel, exhibits an increased sensitivity to GABA. These results suggest that the Hco-UNC-49B subunit is the key determinant for the high agonist sensitivity of heteromeric channels.

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Effect of chronic mild stress and imipramine on the proteome of the rat dentate gyrus.

Publication Date: 2010 Feb 23 PMID: 20180829
Authors: Kedracka-Krok, S. - Fic, E. - Jaciuk, M. - Jankowska, U. - Gruca, P. - Papp, M. - Kusmider, M. - Solich, J. - Debski, J. - Dadlez, M. - Dziedzicka-Wasylewska, M.
Journal: J Neurochem

The present study uses a proteomic approach to examine possible alterations of protein expression in the hippocampus of rats that are subjected to chronic mild stress (CMS). These rats serve as an animal model that was developed to mimic anhedonia, which is one of the core symptoms of depression. Since antidepressant treatment is effective after a few weeks of administration, we also aimed to identify changes that were linked to chronic (once daily for four weeks) and "pulse" (once a week) administration of imipramine (IMI). Fifteen differential proteins were identified with 2D electrophoresis followed by mass spectrometry. Although both methods of imipramine administration restored normal sucrose consumption in rats that were subjected to CMS, the molecular mechanisms of these two therapies were different. CMS-induced changes in the levels of dynactin 2, Ash 2, non-neuronal SNAP25 and alpha-enolase were reversed by chronic imipramine, but "pulse" treatment was not that effective.

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FoxP1 promotes midbrain identity in embryonic stem cell derived dopamine neurons by regulating Pitx3.

Publication Date: 2010 Feb 19 PMID: 20175877
Authors: Konstantoulas, C. J. - Parmar, M. - Li, M.
Journal: J Neurochem

The robust generation of midbrain dopamine neurons from embryonic stem cells and patient specific induced pluripotent stem cells is a prospective tool for the development of new drugs and cell based therapies, and investigations into the aetiology of Parkinson's disease. To achieve this, it is crucial to identify the fate determining regulatory factors that influence dopamine cell fate decision and the underlying molecular machinery. We identified FoxP1 as a novel marker for midbrain dopamine neurons. Enforced expression of FoxP1 in embryonic stem cells actuates the expression of Pitx3, a homeobox protein that is exclusively expressed in midbrain dopaminergic neurons and is required for their differentiation and survival during development and from embryonic stem cells in-vitro. We show that FoxP1 can be recruited to the Pitx3 locus in embryonic stem cells and regulate Pitx3 promoter activity in a dual-luciferase assay. This transcriptional regulation of Pitx3 by FoxP1 depends on the presence of two high affinity binding sites in the distal Pitx3 promoter, through which FoxP1 directly binds as demonstrated by chromatin immunoprecipitation and electrophoretic mobility shift assay. Thus, our study demonstrates for the first time a transcription regulatory role for FoxP1 on the Pitx3 gene in mammalian stem cells.

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Modulation of A1 adenosine receptor expression by cell aggregation and long-term activation of A2a receptors in cultures of avian retinal cells: Involvement of the cyclic AMP/PKA pathway.

Publication Date: 2010 Feb 15 PMID: 20163523
Authors: Pereira, M. R. - Hang, V. R. - Vardiero, E. - de Mello, F. G. - Paes-de-Carvalho, R.
Journal: J Neurochem

Abstract The expression of A1 and A2a adenosine receptors is developmentally regulated in the chick retina, but little is known about the factors important for this regulation. Here we show that cell aggregation and cyclic AMP analogs promote a dramatic increase in A1 receptor expression. Importantly, a long-term stimulation of A2a receptors also promotes an increase of A1 receptor expression accompanied by a down-regulation of A2a receptors. Chick embryo retina cultures grown in the form of aggregates or dispersed cells accumulate cyclic AMP when stimulated with dopamine or the adenosine agonist 2-chloroadenosine. However, inhibition of dopamine-dependent cyclic AMP accumulation by 2-chloroadenosine was observed in aggregate cultures but not in dispersed cell cultures. Accordingly, A1 receptor binding sites were detected in aggregate cultures, but were low or absent from dispersed cell cultures. Interestingly, an increase of A1 binding sites was detected when dispersed cell cultures were treated for 5 days with permeable cyclic AMP analogs, the adenylyl cyclase activator forskolin or A2a receptor agonists. Although a significant amount of A1 receptor protein was detected in dispersed cell cultures by western blot or immunocytochemistry, the long-term stimulation of A2a receptors also promoted an increase of the A1 receptor protein and mRNA, indicating that A2a receptors and cyclic AMP were regulating transcription and/or translation of A1 receptors. We also found an increase of A1 receptors in locations in or near the membrane after treatment with A2a agonist. The long-term stimulation of retinal explants with A2a agonist also promoted an increase of A1 receptor protein. The results indicate that A2a receptors and the cyclic AMP/PKA pathway are involved in the regulation of A1 receptor expression during retinal development.

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TRPM3 is expressed in sphingosine-responsive myelinating oligodendrocytes.

Publication Date: 2010 Feb 15 PMID: 20163522
Authors: Hoffmann, A. - Grimm, C. - Kraft, R. - Goldbaum, O. - Wrede, A. - Nolte, C. - Hanisch, U. K. - Richter-Landsberg, C. - Bruck, W. - Kettenmann, H. - Harteneck, C.
Journal: J Neurochem

Abstract Oligodendrocytes are the myelin-forming cells of the CNS and guarantee proper nerve conduction. Sphingosine, one major component of myelin, has recently been identified to activate TRPM3, a member of the melastatin-related subfamily of TRP (transient receptor potential) channels. TRPM3 has been demonstrated to be expressed in brain with unknown cellular distribution. Here, we show for the first time that TRPM3 is expressed in oligodendrocytes in vitro and in vivo. TRPM3 is present during oligodendrocyte differentiation. Immunohistochemistry of adult rat brain slices revealed staining of white matter areas, which co-localized with oligodendrocyte markers. Analysis of the developmental distribution revealed that, prior to myelination, TRPM3 channels are localized on neurons. On oligodendrocytes they are found after the onset of myelination. RT-PCR studies showed that the transcription of TRPM3 splice variants is also developmentally regulated in vitro. Ca(2+) imaging approaches revealed the presence of a sphingosine-induced Ca(2+) entry mechanism in oligodendrocytes - with a pharmacologic profile similar to the profile published for heterologously expressed TRPM3. These findings indicate that TRPM3 participates as a Ca(2+)-permeable and sphingosine-activated channel in oligodendrocyte differentiation and CNS myelination.

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Chronic in vitro ketosis is neuroprotective but not anticonvulsant.

Publication Date: 2010 Feb 15 PMID: 20163521
Authors: Samoilova, M. - Weisspapir, M. - Abdelmalik, P. - Velumian, A. A. - Carlen, P. L.
Journal: J Neurochem

Abstract The ketogenic diet (KD), used successfully to treat a variety of epilepsy syndromes in humans and to attenuate seizures in different animal models, also provides powerful neuroprotection in various CNS injury models. Yet, a direct role for ketone bodies in limiting seizure and neuronal damage remains poorly understood. Using organotypic hippocampal slice cultures, we established an in vitro model of chronic ketosis for parallel studies of its neuroprotective and anticonvulsant effects. Chronic in vitro treatment with a ketone body, D-beta-hydroxybutyrate (DbetaHB), protected the cultures against chronic hypoglycemia, oxygen-glucose deprivation and NMDA-induced excitotoxicity, but failed to suppress intrinsic and induced seizure-like activity, indicating improved neuroprotection is not directly translated into seizure control. However, chronic in vitro ketosis abolished hippocampal network hyperexcitability following a metabolic insult, hypoxia, demonstrating for the first time a direct link between metabolic resistance and better control of excessive, synchronous, abnormal electrical activity. These findings suggest that the KD and, possibly, exogenous ketone administration, can be more beneficial for treatment of seizures associated with metabolic stress or underlying metabolic abnormalities, and can potentially be used to optimize clinical applications of the traditional KD or its variants.

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Nanomolar vitamin E alpha-tocotrienol inhibits glutamate-induced activation of phospholipase A2 and causes neuroprotection.

Publication Date: 2010 Mar PMID: 20028458
Authors: Khanna, S. - Parinandi, N. L. - Kotha, S. R. - Roy, S. - Rink, C. - Bibus, D. - Sen, C. K.
Journal: J Neurochem

Our previous works have elucidated that the 12-lipoxygenase pathway is directly implicated in glutamate-induced neural cell death, and that such that toxicity is prevented by nM concentrations of the natural vitamin E alpha-tocotrienol (TCT). In the current study we tested the hypothesis that phospholipase A(2) (PLA(2)) activity is sensitive to glutamate and mobilizes arachidonic acid (AA), a substrate for 12-lipoxygenase. Furthermore, we examined whether TCT regulates glutamate-inducible PLA(2) activity in neural cells. Glutamate challenge induced the release of [(3)H]AA from HT4 neural cells. Such response was attenuated by calcium chelators (EGTA and BAPTA), cytosolic PLA(2) (cPLA(2))-specific inhibitor (AACOCF(3)) as well as TCT at 250 nM. Glutamate also caused the elevation of free polyunsaturated fatty acid (AA and docosahexaenoic acid) levels and disappearance of phospholipid-esterified AA in neural cells. Furthermore, glutamate induced a time-dependent translocation and enhanced serine phosphorylation of cPLA(2) in the cells. These effects of glutamate on fatty acid levels and on cPLA(2) were significantly attenuated by nM TCT. The observations that AACOCF(3), transient knock-down of cPLA(2) as well as TCT significantly protected against the glutamate-induced death of neural cells implicate cPLA(2) as a TCT-sensitive mediator of glutamate induced neural cell death. This work presents first evidence recognizing glutamate-induced changes in cPLA(2) as a novel mechanism responsible for neuroprotection observed in response to nanomolar concentrations of TCT.

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Nicotine self-administration diminishes stress-induced norepinephrine secretion but augments adrenergic-responsiveness in the hypothalamic paraventricular nucleus and enhances adrenocorticotropic hormone and corticosterone release.

Publication Date: 2010 Mar PMID: 20028457
Authors: Yu, G. - Sharp, B. M.
Journal: J Neurochem

Chronic nicotine self-administration augments the thalamo-pituitary-adrenal (HPA) responses to stress. Altered neuropeptide expression within corticotropin-releasing factor (CRF) neurons in the hypothalamic paraventricular nucleus (PVN) contributes to this enhanced HPA response to stress. Herein, we determined the role of norepinephrine, a primary regulator of CRF neurons, in the responses to footshock during nicotine self-administration. On day 12-15 of self-administration, microdialysis showed nicotine reduced PVN norepinephrine release by footshock (< 50% of saline). Yet, the reduction in footshock-induced adrenocorticotropic hormone and corticosterone secretion because of intra-PVN prazosin (alpha(1) adrenergic antagonist) was significantly greater in rats self-administering nicotine (2-fold) than saline. Additionally, PVN phenylephrine (alpha(1) agonist) stimulated adrenocorticotropic hormone and corticosterone release to a similar extent in unstressed rats self-administering nicotine or saline. Nicotine self-administration also decreased footshock-induced c-Fos expression in the nucleus of the solitary tract-A2/C2 catecholaminergic neurons that project to the PVN. Therefore, footshock-induced nucleus of the solitary tract activation and PVN norepinephrine input are both attenuated by nicotine self-administration, yet PVN CRF neurons are more responsive to alpha(1) stimulation, but only during stress. This plasticity in noradrenergic regulation of PVN CRF neurons provides a new mechanism contributing to the HPA sensitization to stress by nicotine self-administration and smoking.

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Plumbagin, a novel Nrf2/ARE activator, protects against cerebral ischemia.

Publication Date: 2010 Mar PMID: 20028456
Authors: Son, T. G. - Camandola, S. - Arumugam, T. V. - Cutler, R. G. - Telljohann, R. S. - Mughal, M. R. - Moore, T. A. - Luo, W. - Yu, Q. S. - Johnson, D. A. - Johnson, J. A. - Greig, N. H. - Mattson, M. P.
Journal: J Neurochem

Many phytochemicals function as noxious agents that protect plants against insects and other damaging organisms. However, at subtoxic doses, the same phytochemicals may activate adaptive cellular stress response pathways that can protect cells against a variety of adverse conditions. We screened a panel of botanical pesticides using cultured human and rodent neuronal cell models, and identified plumbagin as a novel potent activator of the nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway. In vitro, plumbagin increases nuclear localization and transcriptional activity of Nrf2, and induces the expression of the Nrf2/ARE-dependent genes, such as heme oxygenase 1 in human neuroblastoma cells. Plumbagin specifically activates the Nrf2/ARE pathway in primary mixed cultures from ARE-human placental alkaline phosphatase reporter mice. Exposure of neuroblastoma cells and primary cortical neurons to plumbagin provides protection against subsequent oxidative and metabolic insults. The neuroprotective effects of plumbagin are abolished by RNA interference-mediated knockdown of Nrf2 expression. In vivo, administration of plumbagin significantly reduces the amount of brain damage and ameliorates-associated neurological deficits in a mouse model of focal ischemic stroke. Our findings establish precedence for the identification and characterization of neuroprotective phytochemicals based upon their ability to activate adaptive cellular stress response pathways.

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Central and peripheral consequences of the chronic blockade of CB1 cannabinoid receptor with rimonabant or taranabant.

Publication Date: 2010 Mar PMID: 20028452
Authors: Martin-Garcia, E. - Burokas, A. - Martin, M. - Berrendero, F. - Rubi, B. - Kiesselbach, C. - Heyne, A. - Gispert, J. D. - Millan, O. - Maldonado, R.
Journal: J Neurochem

The endocannabinoid system plays a crucial role in the pathophysiology of obesity. However, the clinical use of cannabinoid antagonists has been recently stopped because of its central side-effects. The aim of this study was to compare the effects of a chronic treatment with the CB(1) cannabinoid antagonist rimonabant or the CB(1) inverse agonist taranabant in diet-induced obese female rats to clarify the biological consequences of CB(1) blockade at central and peripheral levels. As expected, chronic treatment with rimonabant and taranabant reduced body weight and fat content. Interestingly, a decrease in the number of CB(1) receptors and its functional activity was observed in all the brain areas investigated after chronic taranabant treatment in both lean and obese rats. In contrast, chronic treatment with rimonabant did not modify the density of CB(1) cannabinoid receptor binding, and decreased its functional activity to a lower degree than taranabant. Six weeks after rimonabant and taranabant withdrawal, CB(1) receptor density and activity recovered to basal levels. These results reveal differential adaptive changes in CB(1) cannabinoid receptors after chronic treatment with rimonabant and taranabant that could be related to the central side-effects reported with the use of these cannabinoid antagonists.

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Pathogenic cysteine mutations affect progranulin function and production of mature granulins.

Publication Date: 2010 Mar PMID: 20028451
Authors: Wang, J. - Van Damme, P. - Cruchaga, C. - Gitcho, M. A. - Vidal, J. M. - Seijo-Martinez, M. - Wang, L. - Wu, J. Y. - Robberecht, W. - Goate, A.
Journal: J Neurochem

Frontotemporal dementia with ubiquitin-positive inclusions (FTLD-U) can be caused by mutations in the progranulin gene (GRN). Progranulin (PGRN) is a cysteine-rich growth factor, which is proteolytically cleaved by elastase to produce several granulins (GRNs). All FTLD-U mutations in GRN characterized to date result in reduced secreted PGRN protein. We recently reported a Spanish family with progressive non-fluent aphasia and dementia in which a novel C521Y mutation segregates with disease. A second cysteine mutation (C139R) has also been reported to be disease specific. Allele-specific mRNA expression assays in brain reveal that the C521Y mutant allele is expressed at similar levels to the wild-type allele. Furthermore, plasma PGRN levels in C521Y carriers are comparable with non-carrier family relatives, suggesting that the mutation does not affect PGRN protein expression and secretion in vivo. Despite normal PGRN levels C521Y and C139R mutant GRNs show reduced neurite growth-stimulating activity in vitro. Further study revealed that these mutations also cause impaired cleavage of PGRN by elastase. Our data suggest that these mutations affect the function of full-length PGRN as well as elastase cleavage of PGRN into GRNs, leading to neurodegeneration.

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Involvement of the AT1 receptor subtype in the effects of angiotensin IV and LVV-haemorphin 7 on hippocampal neurotransmitter levels and spatial working memory.

Publication Date: 2010 Mar PMID: 20028450
Authors: De Bundel, D. - Demaegdt, H. - Lahoutte, T. - Caveliers, V. - Kersemans, K. - Ceulemans, A. G. - Vauquelin, G. - Clinckers, R. - Vanderheyden, P. - Michotte, Y. - Smolders, I.
Journal: J Neurochem

Intracerebroventricular (i.c.v.) administration of angiotensin IV (Ang IV) or Leu-Val-Val-haemorphin 7 (LVV-H7) improves memory performance in normal rats and reverses memory deficits in rat models for cognitive impairment. These memory effects were believed to be mediated via the putative 'AT4 receptor'. However, this binding site was identified as insulin-regulated aminopeptidase (IRAP). Correspondingly, Ang IV and LVV-H7 were characterised as IRAP inhibitors. This study investigates whether and how IRAP may be involved in the central effects of Ang IV and LVV-H7. We determined the effects of i.c.v. administration of Ang IV or LVV-H7 on hippocampal neurotransmitter levels using microdialysis in rats. We observed that Ang IV modulates hippocampal acetylcholine levels, whereas LVV-H7 does not. This discrepancy was reflected in the observation that Ang IV binds with micromolar affinity to the AT1 receptor whereas no binding affinity was observed for LVV-H7. Correspondingly, we demonstrated that the AT1 receptor is involved in the effects of Ang IV on hippocampal neurotransmitter levels and on spatial working memory in a plus maze spontaneous alternation task. However, the AT1 receptor was not involved in the spatial memory facilitating effect of LVV-H7. Finally, we demonstrated that Ang IV did not diffuse to the hippocampus following i.c.v. injection, suggesting an extrahippocampal site of action. We propose that AT1 receptors are implicated in the neurochemical and cognitive effects of Ang IV, whereas LVV-H7 may mediate its effects via IRAP.

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Cholesterol modulates ion channels via down-regulation of phosphatidylinositol 4,5-bisphosphate.

Publication Date: 2010 Mar PMID: 20015154
Authors: Chun, Y. S. - Shin, S. - Kim, Y. - Cho, H. - Park, M. K. - Kim, T. W. - Voronov, S. V. - Di Paolo, G. - Suh, B. C. - Chung, S.
Journal: J Neurochem

Ubiquitously expressed Mg(2+)-inhibitory cation (MIC) channels are permeable to Ca2+ and Mg2+ and are essential for cell viability. When membrane cholesterol level was increased by pre-incubating cells with a water-soluble form of cholesterol, the endogenous MIC current in HEK293 cells was negatively regulated. The application of phosphatidylinositol 4,5-bisphosphate (PIP2) recovered MIC current from cholesterol effect. As PIP2 is the direct modulator for MIC channels, high cholesterol content may cause down-regulation of PIP2. To test this possibility, we examined the effect of cholesterol on two exogenously expressed PIP2-sensitive K+ channels: human Ether-a-go-go related gene (HERG) and KCNQ. Enrichment with cholesterol inhibited HERG currents, while inclusion of PIP2 in the pipette solution blocked the cholesterol effect. KCNQ channel was also inhibited by cholesterol. The effects of cholesterol on these channels were blocked by pre-incubating cells with inhibitors for phospholipase C, which may indicate that cholesterol enrichment induces the depletion of PIP2 via phospholipase C activation. Lipid analysis showed that cholesterol enrichment reduced gamma-(32)P incorporation into PIP2 by approximately 35%. Our results suggest that cholesterol may modulate ion channels by changing the levels of PIP2. Thus, an important cross-talk exists among two plasma membrane-enriched lipids, cholesterol and PIP2.

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A novel choline cotransporter sequestration compartment in cholinergic neurons revealed by selective endosomal ablation.

Publication Date: 2010 Mar PMID: 20015153
Authors: Ivy, M. T. - Newkirk, R. F. - Wang, Y. - Townsel, J. G.
Journal: J Neurochem

The sodium-dependent, high affinity choline transporter - choline cotransporter - (ChCoT, aka: cho-1, CHT1, CHT) undergoes constitutive and regulated trafficking between the plasma membrane and cytoplasmic compartments. The pathways and regulatory mechanisms of this trafficking are not well understood. We report herein studies involving selective endosomal ablation to further our understanding of the trafficking of the ChCoT. Selective ablation of early sorting and recycling endosomes resulted in a decrease of approximately 75% of [3H]choline uptake and approximately 70% of [3H]hemicholinium-3 binding. Western blot analysis showed that ablation produced a similar decrease in ChCoTs in the plasma membrane subcellular fraction. The time frame for this loss was approximately 2 h which has been shown to be the constitutive cycling time for ChCoTs in this tissue. Ablation appears to be dependent on the intracellular cycling of transferrin-conjugated horseradish peroxidase and the selective deposition of transferrin-conjugated horseradish peroxidase in early endosomes, both sorting and recycling. Ablated brain slices retained their capacity to recruit via regulated trafficking ChCoTs to the plasma membrane. This recruitment of ChCoTs suggests that the recruitable compartment is distinct from the early endosomes. It will be necessary to do further studies to identify the novel sequestration compartment supportive of the ChCoT regulated trafficking.

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C38, equivalent to BM88, is developmentally expressed in maturing retinal neurons and enhances neuronal maturation.

Publication Date: 2010 Mar PMID: 20002527
Authors: Wakabayashi, T. - Kosaka, J. - Mochii, M. - Miki, Y. - Mori, T. - Takamori, Y. - Yamada, H.
Journal: J Neurochem

C38 antigen is specifically expressed in neuronal cells of the retina. The purpose of this study was to isolate C38 cDNA and determine its molecular functions. Sequence analysis of C38 cDNA revealed that C38 is equivalent to rat BM88, which has been reported to induce cell-cycle arrest and neuronal differentiation in Neuro2a cells. C38 and Ki67, a marker of proliferating cells, were not colocalized during retinal development. C38 was first detected in the retinal ganglion cells at embryonic day 16, much later than the expression of doublecortin, a marker of immature neurons. Although all the horizontal cells were post-mitotic at this stage, C38 was not detected in horizontal cells until the postnatal period. In addition, C38 over-expression did not induce neuronal differentiation or cell-cycle arrest of pluripotent P19 embryonal carcinoma cells. Instead, C38 promoted maturation during neuronal differentiation of P19 embryonal carcinoma cells by down-regulating Oct-3, a pluripotent cell marker and enhancing the expressions of positive regulators of neurogenesis. In conclusion, during retinal development, C38 is first expressed in post-mitotic retinal neurons and is up-regulated during their maturation. C38 does not induce neuronal competence in pluripotent cells, but does promote maturation in already committed neuronal cells.

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Aging-dependent changes of microglial cells and their relevance for neurodegenerative disorders.

Publication Date: 2010 Mar PMID: 20002526
Authors: von Bernhardi, R. - Tichauer, J. E. - Eugenin, J.
Journal: J Neurochem

Among multiple structural and functional brain changes, aging is accompanied by an increase of inflammatory signaling in the nervous system as well as a dysfunction of the immune system elsewhere. Although the long-held view that aging involves neurocognitive impairment is now dismissed, aging is a major risk factor for neurodegenerative diseases such as Alzheimer;s disease, Parkinson;s disease and Huntington's disease, among others. There are many age-related changes affecting the brain, contributing both to certain declining in function and increased frailty, which could singly and collectively affect neuronal viability and vulnerability. Among those changes, both inflammatory responses in aged brains and the altered regulation of toll like receptors, which appears to be relevant for understanding susceptibility to neurodegenerative processes, are linked to pathogenic mechanisms of several diseases. Here, we review how aging and pro-inflammatory environment could modulate microglial phenotype and its reactivity and contribute to the genesis of neurodegenerative processes. Data support our idea that age-related microglial cell changes, by inducing cytotoxicity in contrast to neuroprotection, could contribute to the onset of neurodegenerative changes. This view can have important implications for the development of new therapeutic approaches.

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DNA methylation regulates adenosine A(2A) receptor cell surface expression levels.

Publication Date: 2010 Mar PMID: 20002525
Authors: Buira, S. P. - Albasanz, J. L. - Dentesano, G. - Moreno, J. - Martin, M. - Ferrer, I. - Barrachina, M.
Journal: J Neurochem

Adenosine A(2A) receptors (A(2A)Rs) appear to play important roles in inflammation and in certain diseases of the nervous system. Pharmacological modulation of A(2A)Rs is particularly useful in Parkinson's disease and has been tested in schizophrenia. However, little is known about the regulation of A(2A)R gene (ADORA2A). A bioinformatic analysis revealed the presence of three CpG islands in the 5' UTR region of human ADORA2A. Next, HeLa, SH-SY5Y and U87-MG cells were treated for 48 h with 5 muM 5-azacytidine (Aza). Increased A(2A)R levels were demonstrated in HeLa and SH-SY5Y cells when compared with non-treated cells. No modifications were seen in U87-MG cells. The increased A(2A)R mRNA and protein levels were accompanied by a loss of DNA methylation pattern in HeLa and SH-SY5Y cells, as measured with the SEQUENOM MassArray platform. The Aza treatment also reduced the affinity of a methyl-CpG-binding protein for ADORA2A by quantitative chromatin immunoprecipitation in HeLa cells. Interestingly, A(2A)R levels were reduced by S-adenosyl-l-methionine treatment in U87-MG and methyl-CpG-binding protein affinity was increased for ADORA2A by quantitative chromatin immunoprecipitation. Therefore, these results show for the first time that DNA methylation plays a role in ADORA2A transcription and, subsequently, in constitutive A(2A)R cell surface levels.

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Stress and GABA receptors.

Publication Date: 2010 Mar PMID: 20002524
Authors: Skilbeck, K. J. - Johnston, G. A. - Hinton, T.
Journal: J Neurochem

GABA(A) receptors are sensitive to subtle changes in the environment in both early-life and adulthood. These neurochemical responses to stress in adulthood are sex-dependent. Acute stress induces rapid changes in GABA(A) receptors in experimental animals, with the direction of the changes varying according to the sex of the animals and the stress-paradigm studied. These rapid alterations are of particular interest as they provide an example of fast neurotransmitter system plasticity that may be mediated by stress-induced increases in neurosteroids, perhaps via effects on phosphorylation and/or receptor trafficking. Interestingly, some studies have also provided evidence for long-lasting changes in GABA(A) receptors as a result of exposure to stressors in early-life. The short- and long-term stress sensitivity of the GABAergic system implicates GABA(A) receptors in the non-genetic etiology of psychiatric illnesses such as depression and schizophrenia in which stress may be an important factor.

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Inhibition of Abeta aggregation and neurotoxicity by the 39-kDa receptor-associated protein.

Publication Date: 2010 Mar PMID: 20002523
Authors: Kerr, M. L. - Gasperini, R. - Gibbs, M. E. - Hou, X. - Shepherd, C. E. - Strickland, D. K. - Foa, L. - Lawen, A. - Small, D. H.
Journal: J Neurochem

Aggregation of beta-amyloid protein (Abeta) to form oligomers is considered to be a key step in generating neurotoxicity in the Alzheimer's disease brain. Agents that bind to Abeta and inhibit oligomerization have been proposed as Alzheimer's disease therapeutics. In this study, we investigated the binding of fluorescein-labeled Abeta(1-42) (FluoAbeta(1-42)) to SH-SY5Y neuroblastoma cells and examined the effect of the 39-kDa receptor-associated protein (RAP), on the Abeta cell interaction. FluoAbeta(1-42) bound to the cells in a punctate pattern. Surprisingly, when RAP was added to the incubations, FluoAbeta(1-42) and RAP were found to be co-localized on the cell surface, suggesting that RAP and Abeta may bind to each other. Experiments using the purified proteins confirmed that a RAP-Abeta complex was stable and resistant to sodium dodecyl sulfate. RAP also inhibited Abeta oligomerization. We next examined whether RAP could inhibit the neurotoxic effects of Abeta. Addition of Abeta(1-42) to SH-SY5Y cells caused an increase in intracellular Ca2+ that was inhibited by treatment of the Abeta peptide with RAP. RAP also blocked an Abeta-induced inhibition of long-term memory consolidation in 1-day-old chicks. This study demonstrates that RAP binds to Abeta and is an inhibitor of the neurotoxic effects of Abeta.

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Endogenous purinergic signaling is required for osmotic volume regulation of retinal glial cells.

Publication Date: 2010 Mar PMID: 20002522
Authors: Wurm, A. - Lipp, S. - Pannicke, T. - Linnertz, R. - Krugel, U. - Schulz, A. - Farber, K. - Zahn, D. - Grosse, J. - Wiedemann, P. - Chen, J. - Schoneberg, T. - Illes, P. - Reichenbach, A. - Bringmann, A.
Journal: J Neurochem

Intense neuronal activity in the sensory retina is associated with a volume increase of neuronal cells (Uckermann et al., J. Neurosci. 2004, 24:10149) and a decrease in the osmolarity of the extracellular space fluid (Dmitriev et al., Vis. Neurosci. 1999, 16:1157). Here, we show the existence of an endogenous purinergic mechanism that prevents hypoosmotic swelling of retinal glial (Muller) cells in mice. In contrast to the cells from wild-type mice, hypoosmotic stress induced rapid swelling of glial cell somata in retinal slices from mice deficient in P2Y(1), adenosine A(1) receptors, or ecto-5'-nucleotidase (CD73). Consistently, glial cell bodies in retinal slices from wild-type mice displayed osmotic swelling when P2Y(1) or A(1) receptors, or CD73, were pharmacologically blocked. Exogenous ATP, UTP, and UDP inhibited glial swelling in retinal slices, while the swelling of isolated glial cells was prevented by ATP but not by UTP or UDP, suggesting that uracil nucleotides indirectly regulate the glial cell volume via activation of neuronal P2Y(4/6) and neuron-to-glia signaling. It is suggested that autocrine/paracrine activation of purinergic receptors and enzymes is crucially involved in the regulation of the glial cell volume.

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The neuroprotective mechanism of 1-(R)-aminoindan, the major metabolite of the anti-parkinsonian drug rasagiline.

Publication Date: 2010 Mar PMID: 20002521
Authors: Bar-Am, O. - Weinreb, O. - Amit, T. - Youdim, M. B.
Journal: J Neurochem

The anti-parkinsonian drug, rasagiline [N-propargyl-1-(R)-aminoindan; Azilect(R)], is a secondary cyclic benzylamine and indane derivative, which provides irreversible, potent monoamine oxidase-B (MAO-B) inhibition and possesses neuroprotective and neurorestorative activities. A prospective clinical trial has shown that rasagiline confers significant symptomatic improvement and demonstrated alterations in Parkinson's disease progression. Rasagiline is primarily metabolized by hepatic cytochrome P-450 to form its major metabolite, 1-(R)-aminoindan, a non-amphetamine, weak reversible MAO-B inhibitor compound. Recent studies indicated the potential neuroprotective effect of 1-(R)-aminoindan, suggesting that it may contribute to the overall neuroprotective and antiapoptotic effects of its parent compound, rasagiline. This review article briefly highlights the molecular mechanisms underlying the neuroprotective properties of the active metabolite of rasagiline, 1-(R)-aminoindan, supporting the valuable potential of rasagiline for disease modification.

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Characterization of Ca2+ signaling pathways in mouse adrenal medullary chromaffin cells.

Publication Date: 2010 Mar PMID: 20002295
Authors: Wu, P. C. - Fann, M. J. - Kao, L. S.
Journal: J Neurochem

In the present study, we characterized the Ca2+ responses and secretions induced by various secretagogues in mouse chromaffin cells. Activation of the acetylcholine receptor (AChR) by carbachol induced a transient intracellular Ca2+ concentration ([Ca2+](i)) increase followed by two phases of [Ca2+](i) decay and a burst of exocytic events. The contribution of the subtypes of AChRs to carbachol-induced responses was examined. Based on the results obtained by stimulating the cells with the nicotinic receptor (nAChR) agonist, 1,1-dimethyl-4-phenylpiperazinium iodide, high K(+) and the effects of thapsigargin, it appears that activation of nAChRs induces an extracellular Ca2+ influx, which in turn activate Ca(2+)-induced Ca2+ release via the ryanodine receptors. Muscarine, a muscarinic receptor (mAChRs) agonist, was found to induce [Ca2+](i) oscillation and sustained catecholamine release, possibly by activation of both the receptor- and store-operated Ca2+ entry pathways. The RT-PCR results showed that mouse chromaffin cells are equipped with messages for multiple subtypes of AChRs, ryanodine receptors and all known components of the receptor- and store-operated Ca2+ entry. Furthermore, results obtained by directly monitoring endoplasmic reticulum (ER) and mitochondrial Ca2+ concentration and by disabling mitochondrial Ca2+ uptake suggest that the ER acts as a Ca2+ source, while the mitochondria acts as a Ca2+ sink. Our results show that both nAChRs and mAChRs contribute to the initial carbachol-induced [Ca2+](i) increase which is further enhanced by the Ca2+ released from the ER mediated by Ca(2+)-induced Ca2+ release and mAChR activation. This information on the Ca2+ signaling pathways should lay a good foundation for future studies using mouse chromaffin cells as a model system.

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Ferroportin is a manganese-responsive protein that decreases manganese cytotoxicity and accumulation.

Publication Date: 2010 Mar PMID: 20002294
Authors: Yin, Z. - Jiang, H. - Lee, E. S. - Ni, M. - Erikson, K. M. - Milatovic, D. - Bowman, A. B. - Aschner, M.
Journal: J Neurochem

Although manganese (Mn) is an essential trace element for human development and growth, chronic exposure to excessive Mn levels can result in psychiatric and motor disturbances, referred to as manganism. However, there are no known mechanism(s) for efflux of excess Mn from mammalian cells. Here, we test the hypothesis that the cytoplasmic iron (Fe) exporter ferroportin (Fpn) may also function as a Mn exporter to attenuate Mn toxicity. Using an inducible human embryonic kidney (HEK293T) cell model, we examined the influence of Fpn expression on Mn-induced cytotoxicity and intracellular Mn concentrations. We found that induction of an Fpn-green fluorescent protein fusion protein in HEK293T cells was cytoprotective against several measures of Mn toxicity, including Mn-induced cell membrane leakage and Mn-induced reductions in glutamate uptake. Fpn-green fluorescent protein mediated cytoprotection correlated with decreased Mn accumulation following Mn exposure. Thus, Fpn expression reduces Mn toxicity concomitant with reduced Mn accumulation. To determine if mammalian cells may utilize Fpn in response to increased intracellular Mn concentrations and toxicity, we assessed endogenous Fpn levels in Mn-exposed HEK293T cells and in mouse brain in vivo. We find that 6 h of Mn exposure in HEK293T cells is associated with a significant increase in Fpn levels. Furthermore, mice exposed to Mn showed an increase in Fpn levels in both the cerebellum and cortex. Collectively, these results indicate that (i) Mn exposure promotes Fpn protein expression, (ii) Fpn expression reduces net Mn accumulation, and (iii) reduces cytotoxicity associated with exposure to this metal.

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Neuropeptidomic analysis establishes a major role for prohormone convertase-2 in neuropeptide biosynthesis.

Publication Date: 2010 Mar PMID: 19968759
Authors: Zhang, X. - Pan, H. - Peng, B. - Steiner, D. F. - Pintar, J. E. - Fricker, L. D.
Journal: J Neurochem

Prohormone convertase 2 (PC2) functions in the generation of neuropeptides from their precursors. A quantitative peptidomics approach was used to evaluate the role of PC2 in the processing of peptides in a variety of brain regions. Altogether, 115 neuropeptides or other peptides derived from secretory pathway proteins were identified. These peptides arise from 28 distinct secretory pathway proteins, including proenkephalin, proopiomelanocortin, prodynorphin, protachykinin A and B, procholecystokinin, and many others. Forty one of the peptides found in wild-type (WT) mice were not detectable in any of the brain regions of PC2 knockout mice, and another 24 peptides were present at levels ranging from 20% to 79% of WT levels. Most of the other peptides were not substantially affected by the mutation, with levels ranging from 80% to 120% of WT levels, and only three peptides were found to increase in one or more brain regions of PC2 knockout mice. Taken together, these results are consistent with a broad role for PC2 in neuropeptide processing, but with functional redundancy for many of the cleavages. Comparison of the cleavage sites affected by the absence of PC2 confirms previous suggestions that sequences with a Trp, Tyr, and/or Pro in the P1' or P2' position are preferentially cleaved by PC2 and not by other enzymes present in the secretory pathway.

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Initial calcium release from intracellular stores followed by calcium dysregulation is linked to secondary axotomy following transient axonal stretch injury.

Publication Date: 2010 Mar PMID: 19968758
Authors: Staal, J. A. - Dickson, T. C. - Gasperini, R. - Liu, Y. - Foa, L. - Vickers, J. C.
Journal: J Neurochem

Acute axonal shear and stretch in the brain induces an evolving form of axonopathy and is a major cause of ongoing motor, cognitive and emotional dysfunction. We have utilized an in vitro model of mild axon bundle stretch injury, in cultured primary cortical neurons, to determine potential early critical cellular alterations leading to secondary axonal degeneration. We determined that transient axonal stretch injury induced an initial acute increase in intracellular calcium, principally derived from intracellular stores, which was followed by a delayed increase in calcium over 48 h post-injury (PI). This progressive and persistent increase in intracellular calcium was also associated with increased frequency of spontaneous calcium fluxes as well as cytoskeletal abnormalities. Additionally, at 48 h post-injury, stretch-injured axon bundles demonstrated filopodia-like sprout formation that preceded secondary axotomy and degeneration. Pharmacological inhibition of the calcium-activated phosphatase, calcineurin, resulted in reduced secondary axotomy (p < 0.05) and increased filopodial sprout length. In summary, these results demonstrate that stretch injury of axons induced an initial substantial release of calcium from intracellular stores with elevated intracellular calcium persisting over 2 days. These long-lasting calcium alterations may provide new insight into the earliest neuronal abnormalities that follow traumatic brain injury as well as the key cellular changes that lead to the development of diffuse axonal injury and secondary degeneration.

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d-Serine enhancement of NMDA receptor-mediated calcium increases in rat retinal ganglion cells.

Publication Date: 2010 Mar PMID: 19968757
Authors: Daniels, B. A. - Baldridge, W. H.
Journal: J Neurochem

NMDA receptor (NMDAR) activation is enhanced by d-serine or glycine acting at a specific binding site. Previous work has shown d-serine enhancement of NMDAR currents in retinal ganglion cells. One of the major functions of most NMDA channels is to permit calcium influx into cells. We show that d-serine enhances glutamate-induced calcium responses in immunopanned retinal ganglion cells. This effect was specific to NMDA receptors as similar results were found with NMDA, but not kainate, and was reduced or blocked by modulators of the NMDAR coagonist binding site. d-Serine and glycine enhanced glutamate-induced calcium responses in a dose-dependent manner and at equimolar concentrations there was no difference in the efficacy of the coagonists. In isolated retinas NMDA-induced calcium responses were enhanced by d-serine coapplication in 46% of ganglion cells. Endogenous d-serine degradation by treatment with d-amino acid oxidase caused a approximately 45% decrease in the NMDA-induced response that could be reversed by coapplication with d-serine. d-Serine and glycine were equally effective in enhancing glutamatergic calcium responses. Endogenous d-serine contributes to NMDAR activation in retinal wholemounts and some but not all retinal ganglion cells may experience saturating levels of d-serine or glycine.

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Neuferricin, a novel extracellular heme-binding protein, promotes neurogenesis.

Publication Date: 2010 Mar PMID: 19968755
Authors: Kimura, I. - Nakayama, Y. - Konishi, M. - Kobayashi, T. - Mori, M. - Ito, M. - Hirasawa, A. - Tsujimoto, G. - Ohta, M. - Itoh, N. - Fujimoto, M.
Journal: J Neurochem

We identified a novel extracellular heme-binding protein and named it neuferricin. The recombinant mouse neuferricin produced in High Five cells was secreted efficiently into the culture medium. Mouse neuferricin mRNA was expressed mainly in the brain at the embryo stage and gradually increased during development. At postnatal stage, it was widely expressed in the brain, heart, adrenal gland, and kidney. Mouse neuferricin has 263 amino acids. It has a cytochrome b5-like heme/steroid-binding domain and appeared to bind hemin because neuferricin solution, but not a solution of neuferricinDeltaHBD (a mutant lacking the heme-binding domain), was tinged with brown and had an absorbance peak at 402 nm. In addition, the experiment with anti-neuferricin antibody using heme-affinity chromatography proved that the endogenous neuferricin detected in the culture medium of Neuro2a cells was associated with hemin. Inhibition of endogenous neuferricin by RNA interference excessively promoted cell survival and proliferation and suppressed neurite outgrowth during the induction of differentiation in Neuro2a cells. Addition of recombinant mouse neuferricin, but not neuferricinDeltaHBD, suppressed survival of Neuro2a cells and rescued from the effects of neuferricin RNAi. In primary cultured mouse neural precursor cells, recombinant mouse neuferricin exhibited the ability to promote neurogenesis. The identification of neuferricin, a novel extracellular heme-binding protein with cytochrome b5-like heme/steroid-binding domain and its neurogenic activity, provide new insights not only into brain development but also the function of heme-binding proteins as extracellular signal transmitters.

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Presence of conspecifics and their odor-impregnated objects reverse stress-decreased neurogenesis in mouse dentate gyrus.

Publication Date: 2010 Mar PMID: 19943847
Authors: Cherng, C. G. - Lin, P. S. - Chuang, J. Y. - Chang, W. T. - Lee, Y. S. - Kao, G. S. - Lai, Y. T. - Yu, L.
Journal: J Neurochem

Stress and corticosterone level are thought to negatively associate with neurogenesis in mammalian brains. Social support can diminish many adverse effects of stress. The present study examined the modulating effect of social support on stress-decreased cell proliferation and neuronal differentiation in a mouse model. A randomly-scheduled foot shock followed by restraint in water was used as a profound stress-provoking regimen. Bromodeoxyuridine (BrdU) staining was used to indicate newly mitotic cells and doublecortin (DCx) staining was used to reveal immature neurons. This stress-provoking regimen rapidly decreased BrdU- and BrdU/DCx-labeled cells in the dentate gyrus. However, such a stress-provoking regimen did not affect the number of these labeled cells in the subventricular zone. Familiar and unfamiliar mice' company throughout the stress regimen completely reversed the stress-decreased cell proliferation and neuronal genesis in the dentate gyrus. Likewise, both odor-familiar (from their home cages) and -unfamiliar (from cages other than their home cages) wooden blocks completely reversed the stress-decreased BrdU/DCx-labeled cells in the dentate gyrus. In contrast, wooden blocks free of any odor and camphor odor alone failed to affect the stress-decreased BrdU- or BrdU/DCx-labeled cells. Finally, we showed that conspecifics or their odors during the stress regimen reversed the stress-decreased cell proliferation and neuronal differentiation in the dentate gyrus via a corticosterone-independent mechanism. We conclude that stress and familiarity distinctively affect neurogenesis in the dentate gyrus and subventricular zone. Conspecific companions or presence of their odors reverse stress-decreased neurogenesis in the dentate gyrus, suggesting that social support during stress exposure may improve neurogenesis-related psychological functions.

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