bluuebunny/crossref_metadata_2025 huggingface dataset:
Heparin Oligosaccharides that Pass the Blood‐Brain Barrier Inhibit β‐Amyloid Precursor Protein Secretion and Heparin Binding to β‐Amyloid Peptide
Authors: Leveugle Béatrice , Ding Wanhong , Laurence Fenart , Dehouck Marie‐Pierre , Scanameo Andrew , Cecchelli Roméo , Fillit Howard
Published: July 2010

Abstract: We have previously demonstrated that full‐length heparin stimulates the synthesis and secretion of β‐amyloid precursor protein (APP) through an amyloidogenic pathway in neuroblastoma cells. In the present study, heparin was chemically depolymerized, and the effect of low‐molecular‐weight (LMW) heparin on APP secretion was investigated. In contrast to full‐length heparin, LMW heparin had no significant effect on APP secretion. However, LMW heparin fragments, especially heparin disaccharides, were able to inhibit efficiently the stimulatory effect of heparin on APP secretion. LMW heparin derivatives also inhibit the binding of heparin to the β‐amyloid peptide (1–28). Using an in vitro model, we further demonstrated the passage of LMW heparin derivatives through the blood‐brain barrier. This study suggests that LMW heparin derivatives or analogues may be effective as therapeutic agents to prevent or slow the process of amyloidogenesis in Alzheimer's disease.
Characterization of a Mouse Serotonin 5‐HT3 Receptor Purified from Mammalian Cells
Authors: Hovius Ruud , Tairi Ana‐Paula , Blasey Horst , Bernard Alain , Lundström Kenneth , Vogel Horst
Published: July 2010

Abstract: A serotonin 5‐HT3 receptor was functionally expressed to high levels and on a large scale in mammalian cells with the Semliki Forest virus system. Conditions were optimized to maximize detergent solubilization of the receptor, while preserving ligand binding activity. An efficient one‐step purification yielding ∼50% of the histidine‐tagged 5‐HT3 receptor was achieved with immobilized metal ion chromatography. The expressed receptor, in both membranes and purified preparations, exhibited wild‐type ligand binding properties, characterized by one class of binding sites. The purity of the receptor was shown by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, yielding a single band at 65 kDa, and was confirmed by the specific ligand binding activity of ∼5 nmol/mg of protein. Deglycosylation of the receptor reduced the estimated relative molecular mass to 49 kDa. The apparent molecular mass of the functional receptor complex was determined by size exclusion chromatography to be 280 kDa, suggesting that the 5‐HT3 receptor is a pentameric homooligomer. The secondary structure of the 5‐HT3 receptor as determined by circular dichroism appeared to consist of mainly α‐helices (50%) and β‐strands (24%), with minor contributions from nonregular structure (9%). The binding of either agonist or antagonist did not alter the secondary structure of the receptor.
Examination of Blood‐Brain Barrier Transferrin Receptor by Confocal Fluorescent Microscopy of Unfixed Isolated Rat Brain Capillaries
Authors: Huwyler Jörg , Pardridge William M.
Published: July 2010

Abstract: Isolated rat brain capillaries were analyzed by confocal microscopy. Fluorescent immunoliposomes bearing the OX26 anti‐transferrin receptor monoclonal antibody were synthesized and incubated with freshly isolated unfixed microvessels to visualize binding to luminal and abluminal membranes of the endothelium. Intactness of the endothelial structure was demonstrated by computer‐aided reconstruction of a series of consecutive optical sections. These results indicate that analysis of unfixed brain capillaries by confocal microscopy offers the possibility of assigning the presence of membrane receptors to either the luminal or the basolateral plasma membrane domain.
Serotonin Neural Adaptations to Ontogenetic Loss of Dopamine Neurons in Rat Brain
Authors: Kostrzewa Richard M. , Reader Tomás A. , Descarries Laurent
Published: July 2010

Abstract: In rat, the neonatal destruction of nigrostriatal dopamine (DA) neurons by intracerebral administration of 6‐hydroxydopamine entails dramatic changes in serotonin (5‐hydroxytryptamine, 5‐HT) as well as DA function. Most striking is the 5‐HT hyperinnervation of the adult neostriatum, associated with increases in density of various 5‐HT receptor subtypes and enhanced neuronal responsiveness to the iontophoretic application of 5‐HT and its 5‐HT1B/2C and 5‐HT2A/2C receptor agonists, m‐chlorophenylpiperazine and iododimethoxyphenylaminopropane. The topographical distribution of these changes is consistent with up‐regulation and/or increased production and transport of 5‐HT1B and 5‐HT2A receptors by the neostriatal projection neurons, as confirmed for the 5‐HT2A receptor in a recent in situ hybridization study. It is interesting that this study has also shown that increases in both 5‐HT2A binding and mRNA level were abolished by chronic pretreatment with the DA agonists, apomorphine and SKF 38393, suggesting a regulatory influence of DA in the expression of this 5‐HT receptor. D1 receptor binding is known to be slightly reduced in the rostral neostriatum of these rats, a down‐regulation apparently imputable to a reduced rate of synthesis of the receptor. In contrast, D2 receptor binding is increased throughout the DA‐denervated and 5‐HT‐hyperinnervated neostriatum, perhaps due to some post‐transcriptional modifications. Stereotyped and motor behaviors induced by systemic treatment with D1 and D2 agonists are markedly enhanced in these rats (behavioral supersensitivity), although priming is commonly required to unmask a latent D1 supersensitivity. In the case of oral activity, however, overt behavioral supersensitivity is induced by D1 as well as D2 agonists. Moreover, there is overt supersensitivity of oral activity in response to the 5‐HT receptor agonist m‐chlorophenylpiperazine, which is presumably imputable to 5‐HT2C receptors and may be demonstrated even in the absence of supersensitivity to D1 receptor agonist. 5‐HT adaptations, therefore, seem to play a role not only in the abnormal spontaneous behavior, but also in the behavioral supersensitivity to 5‐HT as well as DA receptor agonists in these rats.
Cloning and Functional Characterization of a σ Receptor from Rat Brain
Authors: Seth Pankaj , Fei You‐Jun , Li Hui W. , Huang Wei , Leibach Frederick H. , Ganapathy Vadivel
Published: July 2010

Abstract: We have cloned a σ receptor from rat brain and established its functional identity using a heterologous expression system. The cloned cDNA (1,582 bp long) codes for a protein of 223 amino acids that possesses a single putative transmembrane domain. The amino acid sequence of the rat brain σ receptor is highly homologous to that of the σ receptor recently cloned from guinea pig liver and a human placental cell line but is not related to any other known mammalian receptors. When expressed in HeLa cells, the rat brain σ receptor cDNA leads to a two‐ to threefold increase in haloperidol binding, and this cDNA‐induced binding is sensitive to inhibition by several σ receptor‐specific ligands. Kinetic analysis using the heterologous expression system has revealed that the rat brain σ receptor interacts with haloperidol with an apparent dissociation constant (KD) of 3 nM. Functional expression of the cloned rat brain σ receptor in HeLa cells also leads to an increase in the binding of two other σ ligands, namely, (+)‐pentazocine and (+)‐3‐(3‐hydroxyphenyl)‐N‐(1‐propyl)piperidine (PPP). Pharmacological characterization of the cloned rat brain σ receptor reveals that it exhibits severalfold higher affinity for clorgyline than for 1,3‐di(2‐tolyl)guanidine, it interacts with progesterone and testosterone, and its interaction with PPP is markedly enhanced by phenytoin. In addition, transfection of MCF‐7 cells, which do not express type 1 σ receptor mRNA or activity, with the cloned rat brain cDNA leads to the appearance of haloperidol‐sensitive binding of (+)‐pentazocine, a selective type 1 σ receptor ligand. These data show that the cloned rat brain cDNA codes for a functional type 1 σ receptor. Northern blot analysis with poly(A)+ RNA isolated from various rat tissues has indicated that the σ receptor‐specific transcript, 1.6 kb in size, is expressed abundantly in liver and moderately in intestine, kidney, brain, and lung.
Developmental Expression of GABAA Receptor Subunit mRNAs in Individual Hippocampal Neurons In Vitro and In Vivo
Authors: Brooks‐Kayal A. R. , Jin H. , Price M. , Dichter M. A.
Published: July 2010

Abstract: The GABAA receptor is a heterooligomeric protein complex composed of multiple receptor subunits. Developmental changes in the pattern of expression of 11 GABAA receptor subunits in individual rat embryonic hippocampal neurons on days 1–21 in culture and acutely dissociated hippocampal neurons from postnatal day (PND) 5 rat pups were investigated using the technique of single‐cell mRNA amplification. We demonstrate that multiple GABAA receptor subunits are expressed within individual hippocampal neurons, with most cells simultaneously expressing α1, α2, α5, β1, and γ2 mRNAs. Further, relative expression of several GABAA receptor subunit mRNAs changes significantly in embryonic hippocampal neurons during in vitro development, with the relative abundance (compared with β‐actin) of α1, α5, and γ2 mRNAs increasing 2.3‐, 2.7‐, and 3.8‐fold, respectively, from days 1 to 14, and β1 increasing 5‐fold from days 1 to 21. In situ hybridization with antisense digoxigenin‐labeled α1, β1, and γ2 RNA probes demonstrates a similar increase in expression of subunit mRNAs as embryonic hippocampal neurons mature in vitro. Relative abundances of α1, β1, and γ2 subunit mRNAs in acutely dissociated PND 5 hippocampal neurons are also significantly greater than in embryonic day 17 neurons on day 1 in vitro and exceed the peak values seen in cultured neurons on days 14–21, suggesting that GABAA receptor subunit mRNA expression within individual hippocampal neurons follows a similar, if somewhat delayed, developmental pattern in vitro compared with in vivo. These findings suggest that embryonic hippocampal neuronal culture provides a useful model in which to study the developmental regulation of GABAA receptor expression and that developmental changes in GABAA receptor subunit expression may underlie some of the differences in functional properties of GABAA receptors in neonatal and mature hippocampal neurons.
A Cell Culture Model for Androgen Effects in Motor Neurons
Authors: Brooks Brian P. , Merry Diane E. , Paulson Henry L. , Lieberman Andrew P. , Kolson Dennis L. , Fischbeck Kenneth H.
Published: July 2010

Abstract: Androgens are known to alter the morphology, survival, and axonal regeneration of lower motor neurons in vivo. To understand better the molecular mechanisms of androgen action in neurons, we created a model system by stably expressing the human androgen receptor (AR) in motor neuron hybrid cells. Motor neuron hybrid cells express markers consistent with anterior horn cells and can be differentiated into a neuronal phenotype. When differentiated in the presence of androgen, AR‐expressing cells, but not control cells, exhibit a dose‐dependent change in morphology: androgen‐treated cells develop larger cell bodies and broader neuritic processes while continuing to express neuronal markers. In addition, androgen promotes the survival of AR‐expressing cells, but not control cells, under low‐serum conditions. Our results demonstrate a direct trophic effect of androgens on lower motor neurons, mediated through the AR expressed in this population of neurons.
Fatty Acid Transport and Utilization for the Developing Brain
Authors: Edmond John , Higa Tami A. , Korsak Rose A. , Bergner E. Ann , Lee W.‐N. Paul
Published: July 2010

Abstract: To determine the transport and utilization of dietary saturated, monounsaturated, and n‐6 and n‐3 polyunsaturated fatty acids for the developing brain and other organs, artificially reared rat pups were fed a rat milk substitute containing the perdeuterated (each 97 atom% deuterium) fatty acids, i.e., palmitic, stearic, oleic, linoleic, and linolenic, from day 7 after birth to day 14 as previously described. Fatty acids in lipid extracts of the liver, lung, kidney, and brain were analyzed by gas chromatography‐mass spectrometry to determine their content of each of the deuterated fatty acids. The uptake and metabolism of perdeuterated fatty acid lead to the appearance of three distinct groups of isotopomers: the intact perdeuterated, the newly synthesized (with recycled deuterium), and the natural unlabeled fatty acid. The quantification of these isotopomers permits the estimation of uptake and de novo synthesis of these fatty acids. Intact perdeuterated palmitic, stearic, and oleic acids from the diet were found in liver, lung, and kidney, but not in brain. By contrast, perdeuterated linoleic acid was found in all these organs. Isotopomers of fatty acid from de novo synthesis were observed in palmitic, oleic, and stearic acids in all tissues. The highest enrichment of isotopomers with recycled deuterium was found in the brain. The data indicate that, during the brain growth spurt and the prelude to myelination, the major saturated and monounsaturated fatty acids in brain lipids are exclusively produced locally by de novo biosynthesis. Consequently, the n‐6 and n‐3 polyunsaturated fatty acids must be transported and delivered to the brain by highly specific mechanisms.
Regulation of G Protein‐Coupled Receptor Kinase 2 in Brains of Opiate‐Treated Rats and Human Opiate Addicts
Authors: Ozaita Andrés , Escribá Pablo V. , Ventayol Pere , Murga Cristina , Mayor Federico , García‐Sevilla Jesús A.
Published: July 2010

Abstract: The effects of opiate drugs (heroin, morphine, and methadone) on the levels of G protein‐coupled receptor kinase 2 (GRK2) were studied in rat and human brain frontal cortices. The density of brain GRK2 was measured by immunoblot assays in acute and chronic opiate‐treated rats as well as in opiate‐dependent rats after spontaneous or naloxone‐precipitated withdrawal and in human opiate addicts who had died of an opiate overdose. In postmortem brains from human addicts, total GRK2 immunoreactivity was not changed significantly, but the level of the membrane‐associated kinase was modestly but significantly increased (12%) compared with matched controls. In rats treated chronically with morphine or methadone modest increases of the enzyme levels (only significant after methadone) were observed. Acute treatments with morphine and methadone induced dose‐ and time‐dependent increases (8–22%) in total GRK2 concentrations [higher increases were observed for the membrane‐associated enzyme (46%)]. Spontaneous and naloxone‐precipitated withdrawal after chronic morphine or methadone induced a marked up‐regulation in the levels of total GRK2 in the rat frontal cortex (18–25%). These results suggest that GRK2 is involved in the short‐term regulation of μ‐opioid receptors in vivo and that the activity of this regulatory kinase in brain could have a relevant role in opiate tolerance, dependence, and withdrawal.
Regional Distribution of Sonic Hedgehog, Patched, and Smoothened mRNA in the Adult Rat Brain
Authors: Traiffort Elisabeth , Charytoniuk Dorota Anna , Faure Helene , Ruat Martial
Published: July 2010

Abstract: In vertebrates, Sonic Hedgehog (Shh), Desert Hedgehog (Dhh), and Indian Hedgehog (Ihh) genes encode a family of morphogen proteins that are implicated in a wide range of signaling activities, particularly during embryonic development. These secreted proteins are proposed to mediate their effects on target cells by interacting with their putative receptor, Patched (Ptc), and with a seven‐pass transmembrane protein, Smoothened (Smo). However, the roles that these signaling molecules may play in adult tissues, particularly in brain, are not yet clearly defined. Therefore, we investigated the expression of these genes in adult rat tissues. Northern blot analysis revealed expression of Shh, Dhh, and Ihh genes in peripheral tissues, whereas Shh transcript was also identified in brain. It is interesting that northern blot analysis with probes derived from the mouse Ptc and Smo genes revealed the expression of a 7.9‐kb and a 3.7‐kb transcript, respectively, in all brain tissues examined. In situ hybridization experiments using specific digoxigenin‐labeled riboprobes showed expression of Ptc and Smo transcripts in discrete brain areas. Shh‐positive cells were observed in restricted regions of the brain. Within the cerebellum, Shh, Ptc, and Smo transcripts were colocalized in the Purkinje cell layer. These data suggest that, besides its roles in determining cell fate and patterning during embryogenesis, the hedgehog signaling pathway may have also important roles in the adult brain.
Ethanol and Regulation of the NMDA Receptor Subunits in Fetal Cortical Neurons
Authors: Kumari Meena , Ticku Maharaj K.
Published: July 2010

Abstract: Previous studies have shown that chronic ethanol treatment up‐regulates the expression of the N‐methyl‐d‐aspartate (NMDA) receptor number and function both in vitro and in vivo. In vitro chronic ethanol treatment specifically augments mRNA levels of the R2B subunit without altering R1 subunit mRNA levels, although similar treatment results in increased levels of both R1 and R2B polypeptides. To further delineate the molecular mechanisms involved in differential regulation of NMDA receptor subunits by chronic ethanol treatment (50 mM, 5 days), we have determined the mRNA stability of the NMDA R1 and R2B subunits and the transcription rate of the respective genes using mouse fetal cortical neurons. Our observations demonstrated that ethanol stabilized the NMDA R1 mRNA over the time period examined (24 h) without altering the stability of the R2B mRNA. Chronic exposure of fetal cortical neurons to ethanol enhanced the rate of R2B gene transcription approximately twofold. Taken together, these results suggest that up‐regulation of the NMDA receptor number seen in cultured cortical neurons after chronic ethanol treatment is due to the regulation of the NMDA R2B receptor subunit at the transcriptional level and that of the NMDA R1 subunit mainly at the posttranscriptional level.
Suppression of Astroglial Nitric Oxide Synthase Expression by Norepinephrine Results from Decreased NOS‐2 Promoter Activity
Authors: Feinstein Douglas L.
Published: July 2010

Abstract: We previously demonstrated that norepinephrine (NE) inhibits induction of the calcium‐independent isoform of nitric oxide synthase (NOS‐2) in primary rat astrocyte cultures. However, the molecular mechanisms underlying this effect are unknown. In C6 cells and astrocytes, NE suppressed both cytokine‐ and lipopolysaccharide (LPS)‐dependent nitrite accumulation, an index of NOS‐2 activity. NE reduced the steady‐state levels of NOS‐2 mRNA induced by LPS plus cytokines but did not decrease NOS‐2 mRNA stability or inhibit activation or subunit composition of transcription factor nuclear factor κB, which is necessary for NOS‐2 induction. In C6 cells stably transfected with a 1,588‐bp mouse NOS‐2 promoter, NE reduced LPS plus cytokine‐induced reporter gene expression, suggesting inhibition of NOS‐2 promoter activity. In contrast, suppression was lost when a truncated 85‐bp NOS‐2 promoter was used, and in these cells NE potentiated reporter gene expression, alone or in the presence of LPS and cytokines. These results suggest that the suppressive effects of NE are due to modification of transcription factor activity in a region located between −1,588 and −85 of the NOS‐2 promoter and may help explain observations that in some cells cyclic AMP can potentiate, rather than suppress, NOS‐2 expression.
Endomorphin‐Stimulated [35S]GTPγS Binding in Rat Brain: Evidence for Partial Agonist Activity at μ‐Opioid Receptors
Authors: Sim Laura J. , Liu Qixu , Childers Steven R. , Selley Dana E.
Published: July 2010

Abstract: Endomorphin‐1 is a peptide whose binding selectivity suggests a role as an endogenous ligand at μ‐opioid receptors. In the present study, the effect of endomorphin‐1 on μ receptor‐coupled G proteins was compared with that of the μ agonist DAMGO by using agonist‐stimulated [35S]GTPγS binding in rat brain. [35S]GTPγS autoradiography revealed a similar localization of endomorphin‐1 and DAMGO‐stimulated [35S]GTPγS binding in areas including thalamus, caudate‐putamen, amygdala, periaqueductal gray, parabrachial nucleus, and nucleus tractus solitarius. Naloxone blocked endomorphin‐1‐stimulated labeling in all regions examined. Although the distribution of endomorphin‐1‐stimulated [35S]GTPγS binding resembled that of DAMGO, the magnitude of endomorphin‐1‐stimulated binding was significantly lower than that produced by DAMGO. Concentration‐effect curves of endomorphin‐1 and DAMGO in thalamic membranes confirmed that endomorphin‐1 produced only 70% of DAMGO‐stimulated [35S]GTPγS binding. Differences in maximal stimulation of [35S]GTPγS binding between DAMGO and endomorphin‐1 were magnified by increasing GDP concentrations, and saturation analysis of net endomorphin‐1‐stimulated [35S]GTPγS binding revealed a lower apparent Bmax value than that obtained with DAMGO. Endomorphin‐1 also partially antagonized DAMGO stimulation of [35S]GTPγS binding. These results demonstrate that endomorphin‐1 is a partial agonist for G protein activation at the μ‐opioid receptor in brain.
Protection Against β‐Amyloid Toxicity in Primary Neurons by Paclitaxel (Taxol)
Authors: Michaelis M. L. , Ranciat N. , Chen Y. , Bechtel M. , Ragan R. , Hepperle M. , Liu Y. , Georg G.
Published: July 2010

Abstract: Neurofibrillary tangles in Alzheimer's disease contain aggregates of abnormally phosphorylated microtubule‐associated protein τ, indicating that microtubule breakdown is a primary event in the neurodegenerative cascade. Recent studies have shown that addition to neuronal cultures of amyloid peptides found in Alzheimer's leads to abnormal phosphorylation of τ and neurofibrillary pathology. We tested the possibility that the microtubule‐stabilizing drug paclitaxel (Taxol) might protect primary neurons against amyloid‐induced toxicity. Neurons exposed to aggregated amyloid peptides 25–35 and 1–42 became pyknotic with degenerating neurites within 24 h. Treatment of cultures with paclitaxel either 2 h before or 2 h after addition of the peptide prevented these morphological alterations. When numbers of viable cells were determined in cultures exposed to amyloid peptide with or without paclitaxel for 24 or 96 h, the percentage of surviving cells was significantly higher in paclitaxel‐treated cultures, and activation of the apoptosis‐associated protease CPP32 was significantly reduced. These observations indicate that microtubule‐stabilizing drugs may help slow development of the neurofibrillary pathology that leads to the loss of neuronal integrity in Alzheimer's disease.
Disruption by Lithium of Phosphoinositide Signalling in Cerebellar Granule Cells in Primary Culture
Authors: Del Río E. , Shinomura T. , Van Der Kaay J. , Nicholls D. G. , Downes C. P.
Published: July 2010

Abstract: Mild depolarisation (20 mM KCI) synergistically enhances the ability of a muscarinic agonist to activate phosphoinositide turnover and to elevate inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] in cerebellar granule cells in primary culture. The effects of lithium on this intense stimulation of phosphoinositide turnover was studied. Lithium causes depletion of cytoplasmic inositol and phosphoinositides, which results in the inhibition of phosphoinositide turnover within 15 min and the return of Ins(1,4,5)P3 to basal levels at this time. This inhibition could not be reversed by culturing and preincubating cerebellar granule cells in concentrations of inositol similar to those detected in the CSF. Inositol concentrations substantially in excess of those in the CSF not only reversed the effects of lithium on stimulated Ins(1,4,5)P3 levels, but significantly enhanced this level in comparison with stimulation in the absence of lithium. sn‐1,2‐Diacylglycerol elevation during stimulated phosphoinositide turnover was also disrupted by lithium, but in contrast to Ins(1,4,5)3, the presence of lithium resulted in a transient enhancement of the elevation evoked by carbachol plus mild KCI depolarisation, which was reversed by 500 µM inositol, but not by 200 µM inositol. The implications of these phenomena in relation to the mechanism of action of lithium in the treatment of manic depression are discussed.
Expression of the Oligodendrocyte‐Myelin Glycoprotein by Neurons in the Mouse Central Nervous System
Authors: Habib Amyn A. , Marton Linda S. , Allwardt Brenda , Gulcher Jeffrey R. , Mikol Daniel D. , Högnason Thorbergur , Chattopadhyay Naibedya , Stefansson Kári
Published: July 2010

Abstract: The oligodendrocyte‐myelin glycoprotein (OMgp) is a 110‐kDa glycosylphosphatidylinositol‐linked protein that was initially identified as a myelin‐specific protein but whose precise function remains unknown. In this study, immunohistochemistry, western blots, in situ hybridization, and northern blots were used to determine the distribution of OMgp in the mouse brain. OMgp is present in a concentration detectable on western blots in the brains of newborn mice, and its concentration gradually increases until day 24 of life. OMgp mRNA is also present in amounts detectable on northern blots in the brains of newborn mice, and its concentration gradually increases until day 21 of life, after which the concentration diminishes a little. Most of the OMgp in the mouse brain appears to be expressed in diverse groups of neurons, but it is particularly prominent in large projection neurons such as the pyramidal cells of the hippocampus, the Purkinje cells of the cerebellum, motoneurons in the brainstem, and anterior horn cells of the spinal cord. However, OMgp is not confined to these cells and is expressed in cells in the white matter as well. The OMgp gene is placed within an intron of the neurofibromatosis type I gene and on the opposite strand. This organization raises the possibility that there may be a relationship between the functions of the products of the two genes. In support of this possibility, we show that within the mouse CNS OMgp and neurofibromin are expressed in the same cell types.
Lithium Increases Tyrosine Hydroxylase Levels Both In Vivo and In Vitro
Authors: Chen Guang , Yuan Pei‐Xong , Jiang Yi‐Ming , Huang Li‐Dong , Manji Husseini K.
Published: July 2010

Abstract: Lithium, a simple monovalent cation, is the mainstay in the treatment of manic‐depressive illness, but despite extensive research, its mechanism of action remains to be elucidated. Because lithium requires chronic administration for therapeutic efficacy and because its beneficial effects last well beyond its discontinuation, it has been postulated that lithium may exert major effects at the genomic level. We have previously shown that lithium, at therapeutically relevant concentrations, increases gene expression through the activator protein‐1 (AP‐1) transcription factor pathway in vitro. In the present study, we have sought to determine if lithium also increases the expression of endogenous genes known to be regulated by AP‐1 and have therefore investigated the effects of lithium on tyrosine hydroxylase (TH) levels. Male Wistar rats were treated with LiCI for 9 days (subacute) or 4 weeks (chronic), and TH levels were measured in frontal cortex, hippocampus, and striatum using immunoblotting. Chronic (but not subacute) lithium treatment resulted in significant increases in TH levels in rat frontal cortex, hippocampus, and striatum. Lithium (1 mM) also increased TH levels in human SH‐SY5Y neuroblastoma cells in vitro, indicating that lithium increases TH levels in both rodent and human tissues, likely via a direct cellular effect. These effects are compatible with (but likely not exclusively due to) an effect on the DNA binding of the 12‐O‐tetradecanoylphorbol 13‐acetate response element to the AP‐1 family of transcription factors.
Traumatic Brain Injury Down‐Regulates Glial Glutamate Transporter (GLT‐1 and GLAST) Proteins in Rat Brain
Authors: Raghavendra Rao Vemuganti L. , Başkaya Mustafa K. , Doğan Aclan , Rothstein Jeffrey D. , Dempsey Robert J.
Published: July 2010

Abstract: Excess activation of NMDA receptors is felt to participate in secondary neuronal damage after traumatic brain injury (TBI). Increased extracellular glutamate is active in this process and may result from either increased release or decreased reuptake. The two high‐affinity sodium‐dependent glial transporters [glutamate transporter 1 (GLT‐1) and glutamate aspartate transporter (GLAST)] mediate the bulk of glutamate transport. We studied the protein levels of GLT‐1 and GLAST in the brains of rats after controlled cortical impact‐induced TBI. With use of subtype‐specific antibodies, GLT‐1 and GLAST proteins were quantitated by immunoblotting in the ipsilateral and contralateral cortex at 2, 6, 24, 72, and 168 h after the injury. Sham‐operated rats served as control. TBI resulted in a significant decrease in GLT‐1 (by 20–45%; p < 0.05) and GLAST (by 30–50%; p < 0.05) protein levels between 6 and 72 h after the injury. d‐[3H]Aspartate binding also decreased significantly (by 30–50%; p < 0.05) between 6 and 72 h after the injury. Decreased glial glutamate transporter function may contribute to the increased extracellular glutamate that may mediate the excitotoxic neuronal damage after TBI. This is a first report showing altered levels of glutamate transporter proteins after TBI.
High‐Field Proton Magnetic Resonance Spectroscopy of a Swine Model for Axonal Injury
Authors: Cecil Kim M. , Lenkinski Robert E. , Meaney David F. , McIntosh Tracy K. , Smith Douglas H.
Published: July 2010

Abstract: A miniature swine model for diffuse brain injury has recently been developed that replicates the inertial loading conditions associated with rotational acceleration during automotive accidents. The swine model induces diffuse axonal pathology without macroscopic injury such as contusions and hematomas, thus affording a unique opportunity to study axonal injury with noninvasive techniques such as magnetic resonance imaging (MRI) and spectroscopy (MRS). In the present study, we evaluated this diffuse injury model with proton MRS, in vivo, using a high‐field (4.0‐T) MR scanner, since MRS has been demonstrated as a sensitive probe for detecting neurochemical abnormalities. Our study examined a region of the swine brain at timepoints before and after brain injury. Spectroscopic results indicate that N‐acetylaspartate/creatine is diminished by at least 20% in regions of confirmed axonal pathology, whereas conventional MRI did not detect any abnormalities. These findings suggest that MRS has high sensitivity in diagnosing microscopic pathology following diffuse brain injury.
Nascent Astrocyte Particles Differ from Lipoproteins in CSF
Authors: LaDu M. J. , Gilligan S. M. , Lukens J. R. , Cabana V. G. , Reardon C. A. , Van Eldik L. J. , Holtzman D. M.
Published: July 2010

Abstract: Little is known about lipid transport and metabolism in the brain. As a further step toward understanding the origin and function of CNS lipoproteins, we have characterized by size and density fractionation lipoprotein particles from human CSF and primary cultures of rat astrocytes. The fractions were analyzed for esterified and free cholesterol, triglyceride, phospholipid, albumin, and apolipoproteins (apo) E, AI, AII, and J. As determined by lipid and apolipoprotein profiles, gel electrophoresis, and electron microscopy, nascent astrocyte particles contain little core lipid, are primarily discoidal in shape, and contain apoE and apoJ. In contrast, CSF lipoproteins are the size and density of plasma high‐density lipoprotein, contain the core lipid, esterified cholesterol, and are spherical. CSF lipoproteins were heterogeneous in apolipoprotein content with apoE, the most abundant apolipoprotein, localized to the largest particles, apoAI and apoAII localized to progressively smaller particles, and apoJ distributed relatively evenly across particle size. There was substantial loss of protein from both CSF and astrocyte particles after density centrifugation compared with gel‐filtration chromatography. The differences between lipoproteins secreted by astrocytes and present in CSF suggest that in addition to delivery of their constituents to cells, lipoprotein particles secreted within the brain by astrocytes may have the potential to participate in cholesterol clearance, developing a core of esterified cholesterol before reaching the CSF. Study of the functional properties of both astrocyte‐secreted and CSF lipoproteins isolated by techniques that preserve native particle structure may also provide insight into the function of apoE in the pathophysiology of specific neurological diseases such as Alzheimer's disease.
In Vitro Binding Properties in Rat Brain of [3H]Ro 25‐6981, a Potent and Selective Antagonist of NMDA Receptors Containing NR2B Subunits
Authors: Mutel Vincent , Buchy Danièle , Klingelschmidt Agnès , Messer Jürg , Bleuel Zaiga , Kemp John A. , Richards John Grayson
Published: July 2010

Abstract: The in vitro binding of a new subtype‐selective NMDA receptor antagonist, [3H]Ro 25‐6981, to rat brain membranes and sections was characterized. The compound bound to a single site on the membranes with a KD of 3 nM and a Bmax of 1.6 pmol/mg of protein. Specific binding, defined with a new NR2B‐specific antagonist, Ro 04‐5595 [1‐(4‐chlorophenyl)‐2‐methyl‐6‐methoxy‐7‐hydroxy‐1,2,3,4‐tetrahydroisoquinoline], at 10 µM, was fully inhibited by several compounds with the following rank order of affinities—Ro 25‐6981 > CP‐101,606 > Ro 04‐5595 = ifenprodil ≫ eliprodil > haloperidol > spermine > spermidine > MgCl2 > CaCl2—and partially inhibited by competitive glutamate recognition site antagonists. A high density of binding sites was detected, radioautographically, in several layers of the cerebral cortex, in the hippocampus, dentate gyrus, tuberculum olfactorium, caudate putamen, medium densities in the globus pallidus, thalamus, spinal cord dorsal horn, and motoneurons, whereas the cerebellum, pons, and medulla were, with a few exceptions, e.g., locus coeruleus, poorly labeled. Overall, the distribution of [3H]Ro 25‐6981 binding sites correlated well with that of NR2B (but not NR2A) transcripts, revealed by in situ hybridization histochemistry. The high affinity of [3H]Ro 25‐6981 for NR2B‐containing receptors renders this compound the ligand of choice to study the regulation of NR2B‐containing receptor expression.
Effects of a Peptide Analogue of the Amphiphilic Domain of the Common Neurotrophin Receptor on Nerve Growth Factor‐Mediated Motility of Human Neuroblastoma Cells
Authors: Wang Wei , Dostaler Suzanne M. , Lawrance Gail , Ross Gregory M. , Riopelle Richard J. , Dow Kimberly E.
Published: July 2010

Abstract: Exposure of human neuroblastoma cells (IMR‐32) to a peptide mimic of the cytoplasmic amphiphilic domain of the common neurotrophin receptor (p75NTR 367–379) resulted in enhanced nerve growth factor (NGF)‐mediated inhibition of cell invasion in vitro. The peptide also enhanced NGF‐mediated neurite extension and GAP‐43 gene expression but had no effect on NGF‐mediated cell survival. These latter functional effects mimicked influences on NGF‐mediated neurite growth in other trkA‐positive cells as reported previously. NGF‐dependent trkA phosphorylation was significantly enhanced by the presence of the peptide, whereas high‐affinity binding of 125I‐NGF, both NGF receptors mRNA and protein expression, and trkA dimer/monomer ratios were not influenced. The studies suggest that ligand‐mediated trkA activation has differential effects on cell motility phenomena and that the amphiphilic domain of p75NTR has a role in this differential signaling.
Multiple Amphiphysin II Splice Variants Display Differential Clathrin Binding: Identification of Two Distinct Clathrin‐Binding Sites
Authors: Ramjaun Antoine R. , McPherson Peter S.
Published: July 2010

Abstract: Amphiphysin I and II are nerve terminal‐enriched proteins that display src homology 3 domain‐mediated interactions with dynamin and synaptojanin. It has been demonstrated that the amphiphysins also bind to clathrin, and we have proposed that this interaction may help to target synaptojanin and dynamin to sites of synaptic vesicle endocytosis. To understand better this potential functional role, we have begun to characterize clathrin‐amphiphysin interactions. Using PCR from adult human cortex cDNA, we have cloned a number of amphiphysin II splice variants. In in vitro binding assays, the amphiphysin II splice variants display differential clathrin binding and define a 44‐amino acid region mediating the interaction. Amphiphysin II truncation and deletion mutants identify two distinct clathrin‐binding domains within this region: one with the sequence LLDLDFDP, the second with the sequence PWDLW. Both domains are conserved in amphiphysin I, and saturation binding analysis demonstrates that both sites bind clathrin with approximately equal affinity. The elucidation of clathrin as a splice‐specific binding partner for amphiphysin II begins to address the potential functional role(s) for the multiple amphiphysin II splice variants and further supports an important function for clathrin‐amphiphysin interactions in protein targeting during endocytosis.
(−)‐Deprenyl Protects Human Dopaminergic Neuroblastoma SH‐SY5Y Cells from Apoptosis Induced by Peroxynitrite and Nitric Oxide
Authors: Maruyama Wakako , Takahashi Tsutomu , Naoi Makoto
Published: July 2010

Abstract: In Parkinson's disease the cell death of dopamine neurons has been proposed to be mediated by an apoptotic death process, in which nitric oxide may be involved. This article reports the induction of apoptosis by nitric oxide and peroxynitrite in human dopaminergic neuroblastoma SH‐SY5Y cells and the antiapoptotic activity of (−)‐deprenyl. After the cells were treated with a nitric oxide donor, NOR‐4, or a peroxynitrite donor, SIN‐1, DNA damage was quantitatively studied using a single‐cell gel electrophoresis (comet) assay. NOR‐4 and SIN‐1 induced DNA damage dose‐dependently. Cycloheximide and alkaline treatment of the cells prevented the DNA damage, indicating that the damage is apoptotic and that it depends on the intracellular signal transduction. Superoxide dismutase and the antioxidants reduced glutathione and α‐tocopherol protected the cells from the DNA damage. (−)‐Deprenyl protected the cells from the DNA damage induced by nitric oxide or peroxynitrite almost completely. The protection by (−)‐deprenyl was significant even after it was washed from the cells, indicating that (−)‐deprenyl may activate the intracellular system against apoptosis. These results suggest that (−)‐deprenyl or related compounds may be neuroprotective to dopamine neurons through its antiapoptotic activity.
Nitric Oxide Production and Perivascular Tyrosine Nitration Following Focal Ischemia in Neonatal Rat
Authors: Coeroli L. , Renolleau S. , Arnaud S. , Plotkine D. , Cachin N. , Plotkine M. , Ben‐Ari Y. , Charriaut‐Marlangue C.
Published: July 2010

Abstract: Oxygen free radicals and nitric oxide (NO•) have been proposed to be involved in acute CNS injury produced by cerebral ischemia; however, controversy remains regarding how they cause injury. Because superoxide generation is triggered during reperfusion, the cytotoxic oxidant peroxynitrite could be formed, but it is not known if this occurs. Dot blot and immunohistochemistry studies were performed on the magnitude and time course of tyrosine nitration and inducible NO synthase (NOS2) in the postischemic rat pup brain. Neonatal ischemia was induced by permanent left middle cerebral artery occlusion in association with 1‐h occlusion of the left common carotid artery in 7‐day‐old Wistar pups. Nitrotyrosine (NT) immunoreactivity was evident in the blood vessels close to the cortical infarct at 48–72 h of recovery, and T lymphocytes were involved with this production. NOS2 immunoreactivity was seen in neutrophils in the same vessels and in the parenchyma at 72 h of recirculation. Whereas NT staining decreased with time, NOS2‐positive neutrophils could be still detected in arachnoid vessels at 14 days of recirculation. We conclude that perivascular reactions mediated by peroxynitrite are important in the cascade of events that lead to brain oxidative stress in neonatal ischemia. Moreover, NO‐related species may serve as a signaling function instead of directly mediating toxicity.
Host Cell‐Specific Folding of the Neuronal Nicotinic Receptor α8 Subunit
Authors: Cooper Sandra T. , Millar Neil S.
Published: July 2010

Abstract: Heterologous expression of the neuronal nicotinic acetylcholine receptor α8 subunit in cultured mammalian cell lines has revealed that the correct folding of this protein is dependent on the host cell type. The α8 subunit, which is able to form homo‐oligomeric ion channels when expressed in Xenopus oocytes, could be detected in all transfected cell lines by both immunoprecipitation and immunofluorescence microscopy with a monoclonal antibody that recognises a linear epitope. In contrast, the α8 subunit could be detected in some but not in all transfected cell lines with a monoclonal antibody that recognises a conformation‐sensitive epitope or by nicotinic radioligand binding. It is interesting that although correctly folded α8 protein could be detected in transfected rat pituitary (GH4C1) cells, only misfolded α8 protein could be detected in a large subpopulation of transfectants (transient or clonal stable isolates). We have also found that the protein encoded by a chimaeric cDNA (constructed from the N‐terminal region of α8 and the C‐terminal domain of the serotonin 5‐HT3 receptor subunit) is expressed efficiently, and in a conformation that binds α‐bungarotoxin, in all cell types examined. These results, together with previous expression studies with the homo‐oligomeric α7 subunit and hetero‐oligomeric nicotinic receptor subunit combinations, suggest that the cell‐specific folding described here is a phenomenon that may be characteristic of homo‐oligomeric nicotinic receptors.
Homeobox Genes in the Developing Mouse Brain
Authors: Vollmer Jean‐Yves , Clerc Roger G.
Published: July 2010

Abstract: Any list of past and recent findings on vertebrate brain prenatal development would have to include the fundamental roles of homeobox genes, the genes encoding the nuclear regulatory homeodomain proteins. The discovery of homeobox genes and their involvement as master regulatory elements in programing the development of an embryo into a complete adult organism has provided a key to our understanding of ontogenesis. Also, the correlation of mouse developmental mutants and their corresponding human syndromes with mutations in homeobox genes has provided further evidence for the fundamental role of homeobox genes during the vertebrate brain embryonic development. Here, we review the expression patterns and the phenotypes of gene mutations that implicate a large repertoire of mouse homeobox genes in the specification of neuronal functions during brain embryogenesis.
Metabolic Dysfunction in Familial, but Not Sporadic, Amyotrophic Lateral Sclerosis
Authors: Browne Susan E. , Bowling Allen C. , Baik M. Jay , Gurney Mark , Brown Robert H. , Beal M. Flint
Published: July 2010

Abstract: Autosomal dominant familial amyotrophic lateral sclerosis (FALS) is associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Previous studies have implicated the involvement of metabolic dysfunction in ALS pathogenesis. To further investigate the biochemical features of FALS and sporadic ALS (SALS), we examined SOD activity and mitochondrial oxidative phosphorylation enzyme activities in motor cortex (Brodmann area 4), parietal cortex (Brodmann area 40), and cerebellum from control subjects, FALS patients with and without known SOD mutations, SALS patients, and disease controls (Pick's disease, progressive supranuclear palsy, diffuse Lewy body disease). Cytosolic SOD activity, predominantly Cu/Zn SOD, was decreased ∼50% in all regions in FALS patients with SOD mutations but was not significantly altered in other patient groups. Marked increases in complex I and II–III activities were seen in FALS patients with SOD mutations but not in SALS patients. We also measured electron transport chain enzyme activities in a transgenic mouse model of FALS. Complex I activity was significantly increased in the forebrain of 60‐day‐old G93A transgenic mice overexpressing human mutant SOD1, relative to levels in transgenic wild‐type animals, supporting the hypothesis that the motor neuron disorder associated with SOD1 mutations involves a defect in mitochondrial energy metabolism.
Signal Transduction in Neuronal Death
Authors: Tong Liqi , Toliver‐Kinsky Tracy , Taglialatela Giulio , Werrbach‐Perez Karin , Wood Tom , Perez‐Polo J. Regino
Published: July 2010

Abstract: Apoptosis in the nervous system is a necessary event during the development of the nervous system and is also present after genotoxic events, be they chronic as in aging or more acute after trauma and ischemia. Apoptotic events reflect an interplay between intrinsic signaling events that rely on cytokines, neurotransmitters, and growth factors and responses to extrinsic events that increase levels of radical oxygen species. Both intrinsically and extrinsically driven signal‐transduction pathways act via transcription factors that regulate the coordinated timely expression of stress‐response genes as part of a decision‐making process that can commit cells to apoptosis or survival. Here we discuss the role of two transcription factors that participate in apoptosis in the nervous system: the activator protein AP‐1 and nuclear factor κB.
Control of Cholesterol Biosynthesis in Schwann Cells
Authors: Fu Qiang , Goodrum Jeffry F. , Hayes Carol , Hostettler Janell D. , Toews Arrel D. , Morell Pierre
Published: July 2010

Abstract: Cholesterol accounts for over one‐fourth of total myelin lipids. We found that, during development of the rat sciatic nerve, expression of mRNA for hydroxymethylglutaryl‐coenzyme A (HMG‐CoA) reductase, the rate‐limiting enzyme in cholesterol biosynthesis, was up‐regulated in parallel with mRNA for P0, the major structural protein of PNS myelin, and with ceramide galactosyltransferase (CGT), the rate‐limiting enzyme in cerebroside biosynthesis. To help establish the nature of this coordinate regulation of myelin‐related genes, we examined their steady‐state mRNA levels in cultured primary Schwann cells. We also assayed synthesis of cholesterol and cerebroside to distinguish how much control of synthetic activity for these two myelin lipids involved mRNA levels for HMG‐CoA reductase and CGT, and how much involved post‐mRNA control mechanisms. Addition of forskolin to cells cultured in media supplemented with normal calf serum resulted in up‐regulation of P0 and CGT mRNA expression and cerebroside synthesis, without corresponding increases in HMG‐CoA reductase mRNA or cholesterol synthesis. Cholesterol synthesis increased approximately threefold in Schwann cells cultured with lipoprotein‐deficient serum, without any increase in HMG‐CoA reductase mRNA. Furthermore, addition of either serum lipoproteins or 25‐hydroxycholesterol decreased cholesterol synthesis without altering HMG‐CoA reductase mRNA levels. We conclude that, as in other tissues, cholesterol synthesis in Schwann cells is regulated primarily by intracellular sterol levels. Much of this regulation occurs at posttranscriptional levels. Thus, the in vivo coordinate up‐regulation of HMG‐CoA reductase gene expression in myelinating Schwann cells is secondary to intracellular depletion of cholesterol, as it is compartmentalized within the myelin. It is probably not due to coordinate control at the level of mRNA expression.