bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucocorticoids Stimulate Inflammatory 5‐Lipoxygenase Gene Expression and Protein Translocation in the Brain
Authors: Uz Tolga , Dwivedi Yogesh , Savani Paresh D. , Impagnatiello Francesco , Pandey Ghanshyam , Manev Hari
Published: March 2003

Abstract : In the brain, the expression of 5‐lipoxygenase (5‐LO), the enzyme responsible for the synthesis of inflammatory leukotrienes, increases during aging. Antiinflammatory drugs are currently being evaluated for the treatment of aging‐associated neurodegenerative diseases such as Alzheimer's disease. Although generally considered antiinflammatory, glucocorticoids, whose production also increases during aging, are not particularly effective in this disease. In human monocytes, 5‐LO mRNA content increases on exposure to the synthetic glucocorticoid dexamethasone, which prompted us to hypothesize that glucocorticoids might increase 5‐LO expression in the brain as well. We treated rats for 10 days either with corticosterone (implanted subcutaneously) or with dexamethasone (injected daily) ; they were killed on day 10 after pellet implantation or 24 h after the 10th dexamethasone injection. We found increased levels of 5‐LO mRNA and protein in hippocampus and cerebellum of glucocorticoid‐treated rats ; 5‐LO‐activating protein (FLAP) mRNA content was not affected. Using western immunobloting, we also observed the concurrent translocation of 5‐LO protein from cytosol to membrane, an indication of its activation. Thus, glucocorticoid‐mediated up‐regulation of the neuronal 5‐LO pathway may contribute to rendering an aging brain vulnerable to degeneration.
Localization of Na,K‐ATPase α/β Isoforms in Rat Sciatic Nerves : Effect of Diabetes and Fish Oil Treatment
Authors: Gerbi A. , Sennoune S. , Pierre S. , Sampol J. , Raccah D. , Vague Ph. , Maixent J.‐M.
Published: March 2003

Abstract : The localization of the Na,K‐ATPase isoenzymes in sciatic

nerve remains controversial, as well as diabetes‐induced changes in

Na,K‐ATPase isoforms. Some of these changes could be prevented by fish oil

therapy. The aim of this study was to determine by confocal microscopy the

distribution of Na,K‐ATPase isoforms (α1, α2, α3, β1,

and β2) in the sciatic nerve, the changes induced by diabetes, and the

preventive effect of fish oil in diabetic neuropathy. This study was performed

in three groups of rats. In the first two groups, diabetes was induced by

streptozotocin and rats were supplemented daily with fish oil or olive oil at

a dosage of 0.5 g/kg of body weight. The third one was a control group that

was supplemented with olive oil. Five antibodies against specific epitopes of

Na,K‐ATPase isoenzymes were applied to stained dissociated nerve fibers with

fluorescent secondary antibodies. The five isoenzymes were documented in

nonspecific regions, Schwann cells (myelin), and the node of Ranvier. The

localization of the α1, α2, and β1 isoenzymes was not

affected by diabetes. In contrast, diabetes induced a decrease of the α2

subunit (p < 0.05) and an up‐regulation of the β2 subunit

(p < 0.05). These modifications were noted in both regions for α2 and were localized at the myelin domain only for the β2. Fish oil supplementation prevented the diabetes‐induced changes in the α2 subunit with an additional up‐regulation. The β2 subunit was not modified. A phenotypic change similar to nerve injury was induced by diabetes. Fish oil supplementation partially prevented some of these changes.
Subunit Composition of Kv1 Channels in Human CNS
Authors: Coleman Sarah K. , Newcombe Jia , Pryke Jonathan , Dolly J. Oliver
Published: March 2003

Abstract : The α subunits of Shaker‐related K+

channels (Kv1.X) show characteristic distributions in mammalian brain and

restricted coassembly. Despite the functional importance of these

voltage‐sensitive K+ channels and involvement in a number of

diseases, little progress has been achieved in deciphering the subunit

composition of the (α)4(β)4 oligomers occurring in human CNS. Thus, the association of α and β subunits was investigated in cerebral grey and white matter and spinal cord from autopsy samples. Immunoblotting established the presence of Kv1.1, 1.2, and 1.4 in all the tissues, with varying abundance. Sequential immunoprecipitations identified the subunits coassembled. A putative tetramer of Kv1.3/1.4/1.1/1.2 was found in grey matter. Both cerebral white matter and spinal cord contained the heterooligomers Kv1.1/1.4 and Kv1.1/1.2, similar to grey matter, but both lacked Kv1.3 and the Kv1.4/1.2 combination. An apparent Kv1.4 homooligomer was detected in all the samples, whereas only the brain tissue possessed a putative Kv1.2 homomer. In grey matter, Kvβ2.1 was coassociated with the Kv1.1/1.2 combination and Kv1.2 homooligomer. In white matter, Kvβ2.1 was associated with Kv1.2 only, whereas Kvβ1.1 coprecipitated with all the α subunits present. This represents the first description of Kv1 subunit complexes in the human CNS and demonstrates regional variations, indicative of functional specialisation.
Evidence for Oxidative Stress in the Subthalamic Nucleus in Progressive Supranuclear Palsy
Authors: Albers David S. , Augood Sarah J. , Martin Deborach M. , Standaert David G. , Vonsattel Jean Paul G. , Beal M. Flint
Published: March 2003

Abstract : Increased free radical production and oxidative stress have been proposed as pathogenic mechanisms in several neurodegenerative disorders. Free radicals interact with biological macromolecules, such as lipids, which can lead to lipid peroxidation. A well‐established marker of oxidative damage to lipids is malondialdehyde (MDA). We measured tissue MDA levels in the subthalamic nucleus (STN) and cerebellum from 11 progressive supranuclear palsy (PSP) cases and 11 age‐matched control cases using sensitive HPLC techniques. In PSP, a significant increase in tissue MDA levels was observed in the STN when compared with the age‐matched control group. By contrast, no significant difference between tissue MDA content was observed in cerebellar tissue from the same PSP and age‐matched control cases. These results indicate that lipid peroxidation may play a role in the pathogenesis of PSP.
Wnt Signaling Induces GLT‐1 Expression in Rat C6 Glioma Cells
Authors: Palos Teresa P. , Zheng Shuang , Howard Bruce D.
Published: March 2003

Abstract : The regulation of glial and neuronal Na+ ‐dependent glutamate/aspartate transporters is of interest because abnormal glutamate transport may be responsible for certain neurological diseases. Because expression of the Wnt‐1 protooncogene results in induction of the glial‐type glutamate transporter GLAST in PC12 neuron‐like cells, we have evaluated the effect of Wnt‐1‐induced signaling on glutamate transporter expression in rat C6 glioma cells. C6 cells are known normally to express EAAC1, a neuronal glutamate transporter, but not the GLAST or the GLT‐1 glutamate transporter. C6 cells that ectopically expressed Wnt‐1 contained a GLT‐1 RNA species similar in size (> 10 kb) to the GLT‐1 transcript present in rat brain, and they also contained a previously unreported 3.3‐kb GLT‐1 RNA species. Both GLT‐1 RNAs contain large parts of the coding region. However, the 3.3‐kb GLT‐1 species contains at least one small deletion within the coding region. The Wnt‐1‐expressing C6 cells contained little, if any, GLT‐1 protein as determined by immunological techniques. We suggest that one or both of the GLT‐1 RNA species induced by Wnt‐1 either fail to be translated or yield abnormal translation products that are quickly degraded. Wnt‐1‐expressing C6 cells may thus represent a novel in vitro system for studying GLT‐1 transporter expression at the transcriptional and/or posttranscriptional levels.
Characterization of Nucleotide Transport into Rat Brain Synaptic Vesicles
Authors: Gualix Javier , Pintor Jesús , Miras‐Portugal Maria Teresa
Published: March 2003

Abstract : ATP transport to synaptic vesicles from rat brain has been studied using the fluorescent substrate analogue 1,N6‐ethenoadenosine 5′‐triphosphate (ε‐ATP), The increase in intravesicular concentration was time dependent for the first 30 min, ε‐ATP being the most abundant nucleotide. The complexity of the saturation curve indicates the existence of kinetic and allosteric cooperativity in the nucleotide transport, which exhibits various affinity states with K0.5 values of 0.39 ± 0.06 and 3.8 ± 0.1 mM with ε‐ATP as substrate. The Vmax values obtained were 13.5 ± 1.4 pmol · min‐1· mg of protein‐1 for the first curve and 28.3 ± 1.6 pmol · min‐1· mg of protein‐1 considering both components. This kinetic behavior can be explained on the basis of a mnemonic model. The nonhydrolyzable adenine nucleotide analogues adenosine 5′‐O‐3‐(thiotriphosphate), adenosine 5′‐(β,γ‐imino)triphosphate and the diadenosine polyphosphates P1,P3‐di(adenosine)triphosphate, P1,P4‐di(adenosine)tetraphosphate, and P1,P5‐di(adenosine)pentaphosphae the nucleotide transport. The mitochondrial ATP/ADP exchange inhibitor atractyloside, N‐ethylmaleimide, and polysulfonic aromatic compounds such as Evans blue and 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid also inhibit ε‐ATP vesicular transport.
A Calcium/Calmodulin‐Dependent Nitric Oxide Synthase, NMDAR2/3 Receptor Subunits, and Glutamate in the CNS of the Cuttlefish Sepia officinalis
Authors: Palumbo Anna , Di Cosmo Anna , Poli Annarita , Di Cristo Carlo , D'Ischia Marco
Published: March 2003

Abstract Chemical, biochemical, and immunohistochemical evidence is reported demonstrating the presence in the brain of the cuttlefish Sepia officinalis of a Ca2+‐dependent nitric oxide synthase, NMDAR2/3 receptor subunits, and glutamate, occurring in neurons and fibers functionally related to the inking system. Nitric oxide synthase activity was concentrated for the most part in the cytosolic fraction and was masked by other citrulline‐forming enzyme(s). The labile nitric oxide synthase could be partially purified by ammonium sulfate precipitation of tissue extracts, followed by affinity chromatography on 2′, 5′‐ADP‐agarose and calmodulin‐agarose. The resulting activity, immunolabeled at 150 kDa on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis by antibodies to rat neuronal nitric oxide synthase, depended on NADPH and tetrahydro‐L‐biopterin, and was inhibited by NG‐nitro‐L‐arginine. NMDAR2/3 subunit‐immunoreactive proteins migrating at 170 kDa could also be detected in brain extracts, along with glutamate (whole brain : 0.32 ± 0.03 μmol of glutamate/mg of protein ; optic lobes : 0.22 ± 0.04 ; vertical complex : 0.06 ± 0.06 basal lobes : 0.58 ± 0.04 ; brachial lobe : 0.77 ± 0.06 ; pedal lobe : 1.04 ± 0.08 ; palliovisceral lobe : 0.86 ± 0.05). Incubation of intact brains with 1.5 mM glutamate or NMDA or the nitric oxide donor 2‐(N,N‐diethylamino)diazenolate‐2‐oxide caused a fivefold rise in the levels of cyclic GMP, indicating operation of the glutamate‐nitric oxide‐cyclic GMP signaling pathway. Immunohistochemical mapping of Sepia CNS showed specific localization of nitric oxide synthase‐like and NMDAR2/3‐like immunoreactivities in the lateroventral palliovisceral lobe, the visceral lobe, and the pallial and visceral nerves, as well as in the sphincters and wall of the ink sac.
Cell Type‐Specific Expression of the Kv3.1 Gene Is Mediated by a Negative Element in the 5′ Untranslated Region of the Kv3.1 Promoter
Authors: Gan Li , Hahn Sang June , Kaczmarek Leonard K.
Published: March 2003

Abstract : The Kv3.1 potassium channel gene is restrictively expressed in the CNS, and its expression level is especially high in neurons that are able to follow synaptic inputs at high frequencies. To understand the transcriptional mechanisms controlling Kv3.1 expression, we have conducted a functional analysis of the Kv3.1 promoter in various cell lines of different tissue origins and in transgenic mice. Our results suggest that an upstream regulatory fragment coupled with the 5′ untranslated region (UTR) is able to confer tissue‐specific expression in both cell lines and in transgenic mice. Deletion analysis of the regulatory region carried out in cell lines reveals that a strong negatively acting element, uniquely residing in the 5′ UTR (+350 to +158), appears able to confer cell type specificity on both the Kv3.1 promoter and the thymidine kinase promoter in transient transfection assays. A weak cell type‐specific enhancer in the proximal region of the promoter (‐123 to ‐71) also contributes to cell type‐specific expression of the Kv3.1 gene.
Decrease of Glial Fibrillary Acidic Protein in Rat Frontal Cortex Following Aluminum Treatment
Authors: Guo‐Ross Shirley X. , Yang Ellen Y. , Walsh Thomas J. , Bondy Stephen C.
Published: March 2003

Abstract : Aluminum lactate was injected either intraperitoneally or stereotactically into the lateral cerebral ventricles of rats. Rats were killed at various times after treatment, and frontal cortex, hippocampus, and striatum were dissected out. Microtiter plate‐based sandwich ELISA and immunohistochemistry were used to measure the glial fibrillary acidic protein (GFAP) concentration. GFAP levels were significantly decreased in frontal cortex 7 days after a single lateral ventricular injection of aluminum lactate and 14 days following systemic treatment. In contrast, neither hippocampus nor striatum exhibited any significant changes in the content of this astrocytic intermediate filament protein after aluminum treatment. Levels of a predominantly astroglial enzyme, glutamine synthetase, were also selectively reduced in the frontal cortex following intraventricular injection of aluminum. This depression exhibited a regional and temporal specificity similar to that found for GFAP. These results suggest a selective and progressive diminution of astrocytic responsivity in frontal cortex following either systemic or intraventricular aluminum dosing. The depression of GFAP levels reported here, which was found in the rat cerebral cortex 7‐14 days after aluminum treatment in a species that does not form neurofilamentous aggregates, may reflect extended impairment of astrocytic function and suggests that these cells may be the primary targets of aluminum neurotoxicity.
Identification of a Mammalian Homologue of the Fungal Tom70 Mitochondrial Precursor Protein Import Receptor as a Thyroid Mitochondrial Precursor Protein Import Receptor as a Thyroid Hormone‐Regulated Gene in Specific Brain Regions
Authors: Alvarez‐Dolado Manuel , González‐Moreno María , Valencia Alfonso , Zenke Martin , Bernal Juan , Muñoz Alberto
Published: March 2003

Abstract : Thyroid hormone is an important regulator of mammalian brain maturation. By differential display PCR, we isolated a cDNA clone (S2) that is specifically upregulated in the striatum of neonatal hypothyroid rats. S2 was identified as KIAA0719, the first human gene distantly homologous to the fungal Tom70, which encodes a member of the translocase mitochondrial outer membrane complex involved in the import of preproteins into the mitochondria. By northern and in situ hybridization studies, KIAA0719 was found to be up‐regulated in the striatum, nucleus accumbens, and discrete cortical layers of 15‐day‐old hypothyroid rats. In contrast, lower expression was found in the olfactory tubercle, whereas no differences were detected in other brain regions. Significantly, treatment of hypothyroid animals with single injections of thyroxine restored the normal levels of KIAA0719 expression. Moreover, treatment of control animals with thyroxine led to a reduced expression, demonstrating a negative hormonal regulation in vivo. Thus, KIAA0719 gene expression is regulated by thyroid hormone in the neonatal rat brain in a region‐specific fashion. Given the role of the homologous Tom70 gene, the alteration of KIAA0719 expression may contribute to the changes in mitochondrial morphology and physiology caused by hypothyroidism in the developing rat brain.
Staurosporine‐Induced Activation of Caspase‐3 Is Potentiated by Presenilin 1 Familial Alzheimer's Disease Mutations in Human Neuroglioma Cells
Authors: Kovacs Dora M. , Mancini Ronald , Henderson John , Na Sang J. , Schmidt Stephen D. , Kim Tae‐Wan , Tanzi Rudolph E.
Published: March 2003

Abstract : Familial Alzheimer's disease (FAD) mutant forms of presenilin 1 (PS1) and 2 have been shown to sensitize cells to apoptotic cell death. Here we explore the effects of FAD mutant forms of PS1 on caspase activation during apoptosis. We show that caspase activation leads to increased generation of alternative C‐terminal fragments (CTFs) from mutant as compared to wild‐type (wt) PS1. For this purpose, very low expression levels of wt, A246E, L286V, and ΔE10 FAD mutant PS1 proteins in stably transfected human H4 neuroglioma cells were used to avoid artifactual induction of spontaneous apoptosis due to overexpression of PS1. Staurosporine treatment of these cells resulted in increased cell death and up to a 10‐fold increase in caspase‐3 activation in mutant versus wt PS1‐expressing cell lines. Correspondingly, relative levels of caspase‐cleaved PS1 CTFs were increased by five‐to sixfold in the FAD mutant versus wt PS1 cells. Elevated caspase activation and caspase cleavage of FAD mutant PS1 suggest the possibility of either a direct proapoptotic effect of mutant PS1 or interference of mutant PS1 with antiapoptotic effects of wt PS1.
Effects of Peripheral Axotomy on Cholecystokinin Neurotransmission in the Rat Spinal Cord
Authors: Bras João Manuel Antunes , Laporte Anne‐Marie , Benoliel Jean Jacques , Bourgoin Sylvie , Mauborgne Annie , Hamon Michel , Cesselin François , Pohl Michel
Published: March 2003

Abstract : Because cholecystokinin (CCK) acts as a
“functional” endogenous opioid antagonist, it has been proposed
that changes in central CCKergic neurotransmission might account for the
relative resistance of neuropathic pain to the analgesic action of morphine.
This hypothesis was addressed by measuring CCK‐related parameters 2 weeks
after unilateral sciatic nerve section in rats. As expected, significant
decreases (‐25‐38%) in the tissue concentrations and in vitro release of both
substance P and calcitonin gene‐related peptide were noted in the dorsal
quadrant of the lumbar spinal cord on the lesioned side. In contrast, the
tissue levels and in vitro release of CCK were unchanged in the same area in
lesioned rats. Measurements in dorsal root ganglia at L4‐L6 levels revealed no
significant changes in proCCK mRNA after the lesion. However, sciatic nerve
section was associated with a marked ipsilateral increase in both CCK‐B
receptor mRNA levels in these ganglia (+70%) and the autoradiographic labeling
of CCK‐B receptors by [3H]pBC 264 (+160%) in the superficial layers of the lumbar dorsal horn. Up‐regulation of CCK‐B receptors rather than CCK synthesis and release probably contributes to increased spinal CCKergic neurotransmission in neuropathic pain.
Glial Fibrillary Acidic Protein Transcription Responses to Transforming Growth Factor‐β1 and Interleukin‐1β Are Mediated by a Nuclear Factor‐1‐Like Site in the Near‐Upstream Promoter
Authors: Krohn Knut , Rozovsky Irina , Wals Pat , Teter Bruce , Anderson Chris P. , Finch Caleb E.
Published: March 2003

Abstract: Elevated expression of glial fibrillary acidic protein (GFAP) is associated with astrocyte activation during responses to injury in the CNS. Because transforming growth factor‐β1 (TGF‐β1) and interleukin‐1β (IL‐1β) are released during neural responses to injury and because these cytokines also modulate GFAP mRNA levels, it is of interest to define their role in GFAP transcription. The increases of GFAP mRNA in response to TGF‐β1 and decreases in response to IL‐1β were shown to be transcriptionally mediated in rat astrocytes transfected with a luciferase‐reporter construct containing 1.9 kb of 5′‐upstream rat genomic DNA. Constructs containing sequential deletions of the rat GFAP 5′‐upstream promoter identified a short region proximal to the transcription start (‐106 to ‐53 bp) that provides full responses to TGF‐β1 and IL‐1β. This region contains an unusual sequence motif with overlapping nuclear factor‐1 (NF‐1)‐ and nuclear factor‐κB (NF‐κB)‐like binding sites and homology to known TGF‐β response elements. Mutagenesis (3‐bp exchanges) in ‐70 to ‐68 bp blocked the induction of GFAP by TGF‐β1 and the repression by IL‐1β. Gel shift experiments showed that the DNA segment ‐85 to ‐63 bp was bound by a factor(s) in nuclear extracts from astrocytes. The concentrations of these DNA binding factors were increased by treatment of astrocytes with TGF‐β1 and decreased by IL‐1β. Binding of these nuclear factors was blocked by mutation of ‐70 to ‐68 bp. Despite homology to NF‐1 or NF‐κB binding sites in the GFAP promoter at segment ‐79 to ‐67 bp, anti‐NF‐κB or anti‐NF‐1 antibodies did not further retard the gel shift of the nuclear factors/DNA complex. Moreover, astrocytic nuclear proteins do not compete for the specific binding to NF‐1 consensus sequence. Thus, nuclear factors from astrocytes that bind to the ‐85‐ to ‐63‐bp promoter segment might be only distantly related to NF‐1 or NF‐κB. These findings are pertinent to the use of GFAP promoter constructs in transgenic animals, because cis‐acting elements in the GFAP promoter are sensitive to cytokines that may be elaborated in response to expression of transgene products.
Oxidative Stress, Mitochondrial Function, and Acute Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells
Authors: Castilho Roger F. , Ward Manus W. , Nicholls David G.
Published: March 2003

Abstract: On exposure to glutamate, cultured rat cerebellar granule cells undergo a delayed Ca2+ deregulation (DCD), which precedes and predicts cell death. We have previously shown that mitochondria control the sensitivity of the neurons to DCD. Mitochondrial depolarization by rotenone/oligomycin before glutamate addition is strongly neuroprotective, and the indication is therefore that mitochondrial Ca2+ loading leads to a delayed loss of bioenergetic function culminating in DCD and cell death. In this report it is shown that superoxide (O2[UNK]) generation in intact cells, monitored by oxidation of hydroethidine to ethidium, was enhanced by glutamate only when mitochondria were polarized. Production of superoxide was higher in the subset of cells undergoing DCD. In the presence of rotenone and oligomycin, addition of glutamate did not result in increased superoxide generation. Menadione‐generated superoxide enhances the DCD of cells exposed to glutamate; in contrast, glutamate‐induced DCD was potently inhibited by the presence of the cell‐permeant antioxidant manganese(III) tetrakis(4‐benzoic acid) porphyrin. An inverse correlation is observed between the cytoplasmic free Ca2+ maintained in individual cells in the presence of glutamate and the ability of these cells to restore basal Ca2+ when NMDA receptors are inhibited and mitochondrial Ca2+ is released. It is concluded that mitochondrial Ca2+ accumulation and reactive oxygen species each contribute to DCD, probably related to damage to a process controlling Ca2+ efflux from the cell.
Molecular Isoform Distribution and Glycosylation of Acetylcholinesterase Are Altered in Brain and Cerebrospinal Fluid of Patients with Alzheimer’s Disease
Authors: Sáez‐Valero Javier , Sberna Gian , McLean Catriona A. , Small David H.
Published: March 2003

Abstract: The glycosylation of acetylcholinesterase (AChE) in CSF was analyzed by lectin binding. AChE from Alzheimer’s disease (AD) patients was found to bind differently to two lectins, concanavalin A and wheat germ agglutinin, than AChE from controls. As multiple isoforms of AChE are present in both CSF and brain, we examined whether the abnormal glycosylation of AD AChE was due to changes in a specific molecular isoform. Globular amphiphilic dimeric (G2a) and monomeric (G1a) isoforms of AChE were found to be differentially glycosylated in AD CSF. Glycosylation of AChE was also altered in AD frontal cortex but not in cerebellum and was also associated with an increase in the proportion of light (G2 and G1) isoforms. This study demonstrates that the glycosylation of AChE is altered in the AD brain and that changes in AChE glycosylation in AD CSF may reflect changes in the distribution of brain isoforms. The study also suggests that glycosylation of AChE may be a useful diagnostic marker for AD.
Effects of Acute Tryptophan Depletion on Plasma and Cerebrospinal Fluid Tryptophan and 5‐Hydroxyindoleacetic Acid in Normal Volunteers
Authors: Williams W. A. , Shoaf S. E. , Hommer D. , Rawlings R. , Linnoila M.
Published: March 2003

Abstract: Brain serotonin synthesis and metabolism (turnover), as indicated by CSF concentrations of 5‐hydroxyindoleacetic acid (5‐HIAA), may depend on plasma concentrations of the essential amino acid L‐tryptophan (TRP). We investigated the biochemical effects of acute plasma TRP depletion (ATD) in normal volunteers undergoing a 36‐h CSF collection via lumbar drain. Six subjects who were in good health were put on a low‐TRP diet (160 mg/day) 24 h before lumbar puncture; this diet was continued for the first 22 h of the CSF collection. At hour 22, subjects ingested a TRP‐deficient 15‐amino acid drink shown previously to deplete plasma TRP. Total plasma TRP, free plasma TRP, and CSF TRP subsequently decreased 86.3, 86.5, and 92.3%, respectively. CSF 5‐HIAA decreased by 32.8%. Plasma total and free TRP concentrations were both decreased at ∼2 h following ingestion of the TRP‐free amino acid drink and were lowest ∼6 h after ATD; CSF TRP and 5‐HIAA were decreased at 2.5 h and ∼4 h after ATD, respectively. CSF TRP was lowest 8.0 h later. CSF 5‐HIAA continued to decrease 14 h after the TRP‐deficient amino acid drink was given.
Association of the Dystroglycan Complex Isolated from Bovine Brain Synaptosomes with Proteins Involved in Signal Transduction
Authors: Cavaldesi Michaela , Macchia Gianfranco , Barca Stefano , Defilippi Paola , Tarone Guido , Petrucci Tamara C.
Published: March 2003

Abstract: Dystroglycan is a transmembrane heterodimeric complex of α and β subunits that links the extracellular matrix to the cell cytoskeleton. It was originally identified in skeletal muscle, where it anchors dystrophin to the sarcolemma. Dystroglycan is also highly expressed in nonmuscle tissues, including brain. To investigate the molecular interactions of dystroglycan in the CNS, we fractionated a digitonin‐soluble extract from bovine brain synaptosomes by laminin‐affinity chromatography and characterized the protein components. The 120‐kDa α‐dystroglycan was the major 125I‐laminin‐labeled protein detected by overlay assay. This complex, in addition to β‐dystroglycan, was also found to contain Grb2 and focal adhesion kinase p125FAK (FAK). Anti‐FAK antibodies co‐immunoprecipitated Grb2 with FAK. However, no direct interaction between β‐dystroglycan and FAK was detected by co‐precipitation assay. Grb2, an adaptor protein involved in signal transduction and cytoskeleton organization, has been shown to bind β‐dystroglycan. We isolated both FAK and Grb2 from synaptosomal extracts by chromatography on immobilized recombinant β‐dystroglycan. In the CNS, FAK phosphorylation has been linked to membrane depolarization and neurotransmitter receptor activation. At the synapses, the adaptor protein Grb2 may mediate FAK‐β‐dystroglycan interaction, and it may play a role in transferring information between the dystroglycan complex and other signaling pathways.
Cellular Distribution and Developmental Expression of AMP‐Activated Protein Kinase Isoforms in Mouse Central Nervous System
Authors: Turnley Ann M. , Stapleton David , Mann Richard J. , Witters Lee A. , Kemp Bruce E. , Bartlett Perry F.
Published: March 2003

Abstract: The mammalian AMP‐activated protein kinase is a heterotrimeric serine/threonine protein kinase with multiple isoforms for each subunit (α, β, and γ) and is activated under conditions of metabolic stress. It is widely expressed in many tissues, including the brain, although its expression pattern throughout the CNS is unknown. We show that brain mRNA levels for the α2 and β2 subunits were increased between embryonic days 10 and 14, whereas expression of α1, β1, and γ1 subunits was consistent at all ages examined. Immunostaining revealed a mainly neuronal distribution of all isoforms. The α2 catalytic subunit was highly expressed in neurons and activated astrocytes, whereas the α1 catalytic subunit showed low expression in neuropil. The γ1 noncatalytic subunit was highly expressed by neurons, but not by astrocytes. Expression of the β1 and β2 noncatalytic subunits varied, but some neurons, such as granule cells of olfactory bulb, did not express detectable levels of either β isoform. Preferential nuclear localization of the α2, β1, and γ1 subunits suggests new functions of the AMP‐activated protein kinase, and the different expression patterns and cellular localization between the two catalytic subunits α1 and α2 point to different physiological roles.
Serotonin 5‐HT2C Receptor RNA Editing Alters Receptor Basal Activity
Authors: Herrick‐Davis Katharine , Grinde Ellinor , Niswender Colleen M.
Published: March 2003

Abstract : Rat and human serotonin 5‐HT2C receptor

isoforms were evaluated for agonist‐independent activation of inositol

phosphate production in COS‐7 cells. The nonedited isoform

(5‐HT2C‐INI) displayed the greatest basal activity, stimulating

inositol phosphate production fourfold over the fully edited isoform

(5‐HT2C‐VGV). All of the other isoforms tested displayed

intermediate levels of basal activity. Decreasing receptor expression levels

by 50% produced a parallel decrease in basal activity. 5‐HT stimulated

inositol phosphate production twofold over basal levels through the

5‐HT2C‐INI receptor and eightfold over basal levels through the

5‐HT2C‐VGV receptor but produced similar maximal levels of inositol

phosphate. 5‐HT competition for [3H]mesulergine binding to

5‐HT2C‐INI best fit a two‐site analysis with KH

= 7.6 nM and KL = 160 nM, whereas

5‐HT2C‐VGV best fit a one‐site model with Ki =

163 nM. [3H]5‐HT labeled 36% of the total population of

5‐HT2C‐INI receptors labeled by [3H]‐mesulergine but

only 12% of 5‐HT2C‐VGV receptors. [3H]5‐HT

KD values increased from 5.1 nM for

5‐HT2C‐INI to 20 nM for 5‐HT2C‐VGV.

[3H]Mesulergine KD values were the same for

both isoforms. 5‐HT EC50 values for inositol phosphate production

increased from 6.1 nM for 5‐HT2C‐INI to 30 nM for

5‐HT2C‐VGV. These results demonstrate that RNA editing decreases

5‐HT2C receptor basal activity, agonist affinity, and potency, indicating that RNA editing may play a role in regulating serotonergic signal transduction and response to drug therapy.
Characterization of Apelin, the Ligand for the APJ Receptor
Authors: Lee Dennis K. , Cheng Regina , Nguyen Tuan , Fan Theresa , Kariyawasam Anula P. , Liu Yang , Osmond Daniel H. , George Susan R. , O'Dowd Brian F.
Published: March 2003

Abstract: The apelin peptide was recently discovered and demonstrated to be the endogenous ligand for the G protein‐coupled receptor, APJ. A search of the GenBank databases retrieved a rat expressed sequence tag partially encoding the preproapelin sequence. The GenBank search also revealed a human sequence on chromosome Xq25‐26.1, containing the gene encoding preproapelin. We have used the rat sequence to screen a rat brain cDNA library to obtain a cDNA encoding the full‐length open reading frame of rat preproapelin. This cDNA encoded a protein of 77 amino acids, sharing an identity of 82% with human preproapelin. Northern and in situ hybridization analyses revealed both human and rat apelin and APJ to be expressed in the brain and periphery. Both sequence and mRNA expression distribution analyses revealed similarities between apelin and angiotensin II, suggesting they that share related physiological roles. A synthetic apelin peptide was injected intravenously into male Wistar rats, resulting in immediate lowering of both systolic and diastolic blood pressure, which persisted for several minutes. Intraperitoneal apelin injections induced an increase in drinking behavior within the first 30 min after injection, with a return to baseline within 1 h.
In Vitro Expression of N‐Acetyl Aspartate by Oligodendrocytes
Authors: Bhakoo Kishore K. , Pearce Daniel
Published: March 2003

Abstract: Magnetic resonance spectroscopy (MRS) provides a noninvasive means of assessing in vivo tissue biochemistry. N‐Acetyl aspartate (NAA) is a major brain metabolite, and its presence is used increasingly in clinical and experimental MRS studies as a putative neuronal marker. A reduction in NAA levels as assessed by in vivo 1H MRS has been suggested to be indicative of neuronal viability. However, temporal observations of brain pathologies such as multiple sclerosis, mitochondrial encephalopathy with lactic acidosis and stroke‐like episodes (MELAS), and hypothyroidism have shown reversibility in NAA levels, possibly reflecting recovery of neuronal function. A knowledge of the cellular localisation of NAA is critical in interpreting these findings. The assumption that NAA is specific to neurones is based on previous immunohistochemical studies on whole brain using NAA‐specific antibodies. The neuronal localisation was further substantiated by cell culture experiments in which its presence in the oligodendrocyte‐type 2 astrocyte progenitors and immature oligodendrocytes, but not in the mature oligodendrocytes, was observed. More recently, studies on oligodendrocyte biology have revealed the requirement for trophic factors to promote the generation, maturation, and survival of oligodendrocytes in vitro. Here, we have used this new information to implement a more pertinent cell cultivation procedure and demonstrate that mature oligodendrocytes can express NAA in vitro. This observation brings into question whether the NAA changes observed in clinical in vivo 1H MRS studies reflect neuronal function alone. The data presented here support the hypothesis that oligodendrocytes may express NAA in vivo and contribute to the NAA signal observed by 1H MRS.
Phe13, Tyr19‐Melanin‐Concentrating Hormone and the Blood—Brain Barrier
Authors: Kastin Abba J. , Akerstrom Victoria , Hackler Laszlo , Zadina James E.
Published: March 2003

Abstract: Melanin‐concentrating hormone (MCH), found both peripherally and centrally, is involved in food ingestion. Although its expression in brain is increased by fasting, it is not known whether it crosses the bloodbrain barrier (BBB). Use of the sensitive method of multiple‐time regression analysis has shown that almost all of the peptides and polypeptides tested cross the BBB at a rate faster than the vascular marker albumin. With this same method, however, we found that the 19‐amino acid 125I‐Phe13, Tyr19‐MCH did not cross faster than 99mTc‐albumin. Several mechanisms were excluded as possible explanations for the slow rate of influx. These included degradation, association with capillary endothelial cells, and transport from brain to blood. When Phe13, Tyr19‐MCH was perfused in blood‐free buffer, however, it entered the brain significantly faster than albumin. This suggested protein binding as an explanation for the slow rate of influx when the MCH was administered in blood. Protein binding was confirmed by capillary zone electrophoresis, which showed that almost all of the Phe13, Tyr19‐MCH added to blood migrated with a large‐molecular‐weight substance. Sodium dodecyl sulfate—capillary gel electrophoresis of Phe13, Tyr19‐MCH in buffer additionally showed that the MCH aggregated as a trimer, a factor not preventing its influx by blood‐free perfusion. Thus, the results show that blood‐borne Phe13, Tyr19‐MCH does not significantly cross the BBB, probably because of its binding to serum proteins.
Activation of Adensine A1 and A2A Receptors Modulates Dopamine D2 Receptor‐Induced Responses in Stably Transfected Human Neuroblastoma Cells
Authors: Salim Hassan , Ferré Sergi , Dalal Abderrahim , Peterfreund Robert A. , Fuxe Kjell , Vincent Jean‐Didier , Lledo Pierre‐Marie
Published: March 2003

Abstract: Adenosine can influence dopaminergic neuro‐transmission in the basal ganglia via postsynaptic inter‐action between adenosine A2A and dopamine D2 receptors. We have used a human neuroblastoma cell line (SH‐SY5Y) that was found to express constitutively moderate levels of adenosine A1 and A2A receptors (∼100 fmol/mg of protein) to investigate the interactions of A2A/D2 receptors, at a cellular level. After transfection with human D2L receptor cDNA, SH‐SY5Y cells expressed between 500 and 1,100 fmol of D2 receptors/mg of protein. In membrane preparations, stimulation of adenosine A2A receptors decreased the affinity of dopamine D2 receptors for dopamine. In intact cells, the calcium concentration elevation induced by KCl treatment was moderate, and dopamine had no effect on either resting intracellular free Ca2+ concentration ([Ca2+]i) or KCl‐induced responses. In contrast, pretreatment with adenosine deaminase for 2 days dramatically increased the elevation of [Ca2+]i evoked by KCl, which then was totally reversed by dopamine. The effects induced by 48‐h adenosine inactivation were mimicked by application of adenosine A1 antagonists and could not be further reversed by acute activation of either A1 or A2A receptors. Acute application of the selective A2 receptor agonist CGS‐21680 counteracted the D2 receptor‐induced [Ca2+]i responses. The present study shows that SH‐SY5Y cells are endowed with functional adenosine A2A and A1 receptors and that A2A receptors exert an antagonistic acute effect on dopamine D2 receptor‐mediated functions. In contrast, A1 receptors induce a tonic modulatory role on these dopamine functions.
Amyloid β and Amylin Fibrils Induce Increases in Proinflammatory Cytokine and Chemokine Production by THP‐1 Cells and Murine Microglia
Authors: Yates Stephen L. , Burgess Loyd H. , Kocsis‐Angle June , Antal Joyce M. , Dority Michael D. , Embury Paula B. , Piotrkowski Anthony M. , Brunden Kurt R.
Published: March 2003

Abstract: Activated microglia surrounding amyloid β‐containing
senile plaques synthesize interleukin‐1, an inflammatory cytokine that has
been postulated to contribute to Alzheimer's disease pathology. Studies have
demonstrated that amyloid β treatment causes increased cytokine release
in microglia and related cell cultures. The present work evaluates the
specificity of this cellular response by comparing the effects of amyloid
β to that of amylin, another amyloidotic peptide. Both
lipopolysaccharide‐treated THP‐1 monocytes and mouse microglia showed
significant increases in mature interleukin‐1β release 48 h following
amyloid β or human amylin treatment, whereas nonfibrillar rat amylin had
no effect on interleukin‐1β production by THP‐1 cells.
Lipopolysacharide‐stimulated THP‐1 cells treated with amyloid β or amylin
also showed increased release of the proinflamatory cytokines tumor necrosis
factor‐α and interleukin‐6, as well as the chemokines interleukin‐8 and
macrophage inflammatory protein‐1 α and ‐1β. THP‐1 cells incubated
with fibrillar amyloid β or amylin in the absence of lipopolysaccharide
also showed significant increases of both interleukin‐1β and tumor
necrosis factor‐α mRNA. Furthermore, treatment of THP‐1 cells with
amyloid fibrils resulted in an elevated expression of the immediate‐early
genes c‐fos and junB. These studies provide further evidence that fibrillar amyloid peptides can induce signal transduction pathways that initiate an inflammatory response that is likely to contribute to Alzheimer's disease pathology.
Microtubule/MAP‐Affinity Regulating Kinase (MARK) Is Activated by Phenylarsine Oxide In Situ and Phosphorylates Tau Within Its Microtubule‐Binding Domain
Authors: Jenkins Scott M. , Johnson Gail V. W.
Published: March 2003

Abstract: Tau is a microtubule‐associated protein (MAP) that is
functionally modulated by phosphorylation and that is hyperphosphorylated in
several neurodegenerative diseases. Because phosphorylation regulates both
normal and pathological tau functioning, it is of interest to identify the
signaling pathways and enzymes capable of modulating tau phosphorylation in
vivo. Previously, it was demonstrated that in SH‐SY5Y human neuroblastoma
cells and rat primary cortical cultures tau is phosphorylated at
Ser262/356, within its microtubule‐binding domain, by a
staurosporine‐sensitive protein kinase in response to the vicinal
thiol‐directed agent phenylarsine oxide. The current study demonstrates the
presence of a 100‐kDa protein kinase activity in SH‐SY5Y cells that associates
with microtubules, phosphorylates tau at Ser262/356, is activated
by phenylarsine oxide, and is inhibited by the protein kinase inhibitor
staurosporine. Isolation of individual protein bands from a polyacrylamide gel
revealed two closely spaced proteins containing Ser262/356‐directed protein kinase activity. Mass spectrometry analysis indicated that these protein bands correspond to the 100‐kDa microtubule/MAP‐affinity regulating kinase (MARK), which has been shown previously to phosphorylate tau within its microtubule‐binding domain. Immunoblot analysis of the protein kinase bands confirmed this finding, providing the first demonstration that activation of endogenous MARK results in increased tau phosphorylation within its microtubule‐binding domain in situ.
GM3 α2,8‐Sialyltransferase (GD3 Synthase)
Authors: Daniotti Jose L. , Martina Jose A. , Giraudo Claudio G. , Zurita Adolfo R. , Maccioni Hugo J. F.
Published: March 2003

Abstract: GD3 synthase (Sial‐T2) is a key enzyme of ganglioside
synthesis that, in concert with GM2 synthase (GAlNAc‐T), regulates the ratio
of a‐ and b‐pathway gangliosides. In this work, we study the sub‐Golgi
location of an epitope‐tagged version of chicken Sial‐T2 transfected to CHO‐K1
cells. The expressed protein was enzymatically active both in vitro and in
vivo and showed a molecular mass of ∼47 or ∼95 kDa on sodium dodecyl
sulfate‐polyacrylamide gel electrophoresis in the presence or absence of,
respectively, β‐mercaptoethanol. The 95‐kDa form of Sial‐T2 was also
detected if the protein was retained in the endoplasmic reticulum (ER) due to
impaired glycosylation, indicating that it was formed in the ER. Confocal
immunofluorescence microscopy showed Sial‐T2 localized to the Golgi complex
and, within the organelle, partially co‐localizing with the
mannose‐6‐phosphate receptor, a marker of the trans‐Golgi network
(TGN). In cells treated with brefeldin A, a major fraction of Sial‐T2
redistributed to the ER, even under controlled expression to control for
mislocalization due to protein overloading. In experiments of incorporation of
sugars into endogenous acceptors of Golgi membranes in vitro, GD3 molecules
formed by incubation with CMP‐NeuAc were converted to GD2 upon incubation with
UDP‐GalNAc. These results indicate that Sial‐T2 localizes mainly to the
proximal Golgi, although a fraction is located in the TGN functionally coupled
to GalNAc‐T. Consistent with this, most of the enzyme was in an
endoglycosidase H (Endo‐H)‐sensitive, neuraminidase (NANase)‐insensitive form.
A minor secreted form lacking ∼40 amino acids was Endo‐H‐resistant and
NANase‐sensitive, indicating that the cells were able to process
N‐glycans to Endo‐H‐resistant forms. Taken together, the results of these biochemical and immunocytochemical experiments indicate that in CHO‐K1 cells, most Sial‐T2 localizes in the proximal Golgi and that a functional fraction is also present in the TGN.
Cysteine‐String Protein
Authors: Chamberlain Luke H. , Burgoyne Robert D.
Published: March 2003

Abstract: Cysteine‐string protein (Csp) is a major synaptic vesicle and secretory granule protein first discovered in Drosophila and Torpedo. Csps were subsequently identified from Xenopus, Caenorhabditis elegans, and mammalian species. It is clear from the study of a null mutant in Drosophila that Csp is required for viability of the organism and that it has a key role in neurotransmitter release. In addition, other studies have directly implicated Csp in regulated exocytosis in mammalian neuroendocrine and endocrine cell types, and its distribution suggests a general role in regulated exocytosis. An early hypothesis was that Csp functioned in the control of voltage‐gated Ca2+ channels. Csp, however, must have an additional function as a direct regulator of the exocytotic machinery as changes in Csp expression modify the extent of exocytosis triggered directly by Ca2+ in permeabilised cells. Csps possess a cysteine‐string domain that is highly palmitoylated and confers membrane targeting. In addition, Csps have a conserved “J” domain that mediates binding to an activation of the Hsp70/Hsc70 chaperone ATPases. This and other evidence implicate Csps as molecular chaperones in the synapse that are likely to control the correct conformational folding of one or more components of the vesicular exocytotic machinery. Targets for Csp include the vesicle protein VAMP/synaptobrevin and the plasma membrane protein syntaxin 1, the significance of which is discussed in possible models to account for current knowledge of Csp function.
A Functional Analysis of EP4 Receptor‐Expressing Neurons in Mediating the Action of Prostaglandin E2 Within Specific Nuclei of the Brain in Response to Circulating Interleukin‐1β
Authors: Zhang Ji , Rivest Serge
Published: March 2003

Abstract: Proinflammatory cytokines released by cells of myeloid lineage have the ability to stimulate different populations of neurons through intermediate molecules released by cells of the blood‐brain barrier. The aim of the present study was to verify the hypothesis that prostaglandins (PGs) play a site‐specific role in activating selective groups of neurons via a privileged interaction between PG of the E2 type and its EP4 receptor. In a first set of experiments, animals were treated with the inhibitor of PG synthesis ketorolac to determine the endogenous contribution of PG in mediating the neuronal activation and EP4 expression in response to circulating interleukin‐1β (IL‐1β). The subsequent experiment consisted of evaluating the role of PGE2 in activating EP4‐expressing neurons in the rat brain. Ketorolac completely abolished the endogenous release of PGE2 in the liver and prevented the induction of immediate‐early genes and up‐regulation of EP4 mRNA in specific groups of neurons, such as the parvocellular paraventricular nucleus and the A1 catecholaminergic population of cells. This effect was, however, not generalized throughout the brain as PGE2 inhibition failed to abolish IL‐1β‐induced c‐fos transcription in the nucleus of the solitary tract, parabrachial nucleus, bed nucleus of the stria terminalis, and the circumventricular organs. Of interest are the data that central PGE2 injection activated EP4 gene transcription in neurons that no longer responded to the intravenous IL‐1β bolus when the animals were pretreated with ketorolac. Site‐specific interaction between the ligand and its receptor was further supported by the induction of c‐fos‐immunoreactive nuclei within EP4‐expressing neurons in response to intracerebroventricular PGE2 infusion. Both intracerebroventricular PGE2 and intravenous IL‐1β injection provoked a sharp and rapid increase in plasma corticosterone levels, an effect that was completely prevented in inhibiting PG production in IL‐1β‐challenged rats. These data provide the evidence that EP4 is expressed in numerous nuclei involved in autonomic and neuroendocrine control, although a privileged interaction seems to take place in specific nuclei and areas, including the endocrine hypothalamus and the A1 cell group of the ventrolateral medulla. It is quite possible that EP4 acts as the functional receptor for PGE2 to activate the neuronal circuit involved in the activation of the glucocorticoid axis, as an essential neuroendocrine response for the appropriate control of systemic inflammation.