bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Two‐Site Enzyme Immunoassay Reveals the Regional Distributions of and Developmental Changes in GluR1 and NMDAR1 Protein Contents in the Rat Brain
Authors: Ibaraki Kyoko , Otsu Yo , Nawa Hiroyuki
Published: March 2003

Abstract: Glutamate receptors, including the α‐amino‐3‐hydroxy‐4‐methylisoxazole‐4‐propionic acid (AMPA) and NMDA receptors, play an important role in neural development and synaptic plasticity in the brain. To date, it has been difficult to correlate accurately individual biochemical phenomena with quantitative and qualitative changes in receptors occurring in specific neurons or synapses. In the present study, we established a two‐site enzyme immunoassay for two key subunits of the AMPA and NMDA receptors. Its sensitivities were extremely high, 30 pg for GluR1 and 15 pg for the NMDAR1 receptor containing the C2 exon [NMDAR1(C2)], which enabled us to measure their contents in a few milligrams of hippocampal tissue. Regional and developmental variations in receptor protein levels were much more marked than those reported for mRNA: The absolute GluR1 protein content was highest in the rat hippocampus, whereas the NMDAR1(C2) content was high in all the forebrain regions examined. GluR1 protein levels increased most markedly during the second and third weeks of postnatal life, whereas NMDAR1(C2) content increased during the first postnatal week. In the adult rat brain, the ratio of GluR1 protein to NMDAR1 protein was markedly lower in neocortical regions (∼2%) and the highest in cerebellum (22%). Therefore, this two‐site enzyme immunoassay is a specific and unique method that enables us to measure absolute tissue contents of the glutamate receptors and will lead to further important discoveries on the biochemical alterations of these receptors.
1,25‐Dihydroxyvitamin D3 Regulates the Synthesis of γ‐Glutamyl Transpeptidase and Glutathione Levels in Rat Primary Astrocytes
Authors: Garcion E. , Sindji L. , Leblondel G. , Brachet P. , Darcy F.
Published: March 2003

Abstract : Astrocytes play a pivotal role in CNS detoxification

pathways, where glutathione (GSH) is involved in the elimination of oxygen and

nitrogen reactive species such as nitric oxide. We have previously

demonstrated that the specific activity of γ‐glutamyl transpeptidase

(γ‐GT), an enzyme of central significance in GSH metabolism, is

regulated in vivo in astrocytes by 1,25‐dihydroxyvitamin D3

(1,25‐D3). The aim of the present work was to investigate, in

primary cultures of newborn rat astrocytes, the effects of this hormone on

γ‐GT synthesis and on GSH and nitrite levels after lipopolysaccharide

(LPS) treatment. This study demonstrates that both γ‐GT gene expression

and specific activity, induced by LPS, are potentiated by 1,25‐D3.

In contrast, 1,25‐D3 does not regulate the expression of other

enzymes involved in astrocyte detoxification processes, such as superoxide

dismutase or GSH peroxidase. In parallel, 1,25‐D3 enhanced

intracellular GSH pools and significantly reduced nitrite production induced

by LPS. Taken together, these results suggest that γ‐GT, GSH, and

1,25‐D3 play a fundamental role in astrocyte detoxification pathways.
Distribution of Equilibrative, Nitrobenzylthioinosine‐Sensitive Nucleoside Transporters (ENT1) in Brain
Authors: Anderson Christopher M. , Xiong Wei , Geiger Jonathan D. , Young James D. , Cass Carol E. , Baldwin Stephen A. , Parkinson Fiona E.
Published: March 2003

Abstract : Nucleoside transport processes may play a role in

regulating endogenous levels of the inhibitory neuromodulator adenosine in

brain. The cDNAs encoding species homologues of one member of the

equilibrative nucleoside transporter (ENT) gene family have recently been

isolated from rat (rENT1) and human (hENT1) tissues. The current study used

RT‐PCR, northern blot, in situ hybridization, and

[3H]nitrobenzylthioinosine autoradiography to determine the

distribution of mRNA and protein for ENT1 in rat and human brain. Northern

blot analysis indicated that hENT1 mRNA is widely distributed in adult human

brain. 35S‐labeled sense and antisense riboprobes, transcribed from a 153‐bp segment of rENT1, were hybridized to fresh frozen coronal sections from adult rat brain and revealed widespread rENT1 mRNA in pyramidal neurons of the hippocampus, granule neurons of the dentate gyrus, Purkinje and granule neurons of the cerebellum, and cortical and striatal neurons. Regional localization in rat brain was confirmed by RT‐PCR. Thus, ENT1 mRNA has a wide cellular and regional distribution in brain, indicating that this nucleoside transporter subtype may be important in regulating intra‐ and extracellular levels of adenosine in brain.
Calnexin and the Immunoglobulin Binding Protein (BiP) Coimmunoprecipitate with AMPA Receptors
Authors: Rubio Maria E. , Wenthold Robert J.
Published: March 2003

Abstract : To identify proteins that interact with α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptors, we carried out coimmunoprecipitation analyses on detergent‐solubilized rat forebrain membranes. Membranes were solubilized with Triton X‐100, and immunoprecipitation was done using subunit‐specific antibodies to GluR1, GluR2/3, and GluR4 attached to protein A‐agarose. Proteins bound to the antibodies were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis followed by silver staining and western blotting. With solubilization in low ionic strength buffer, several coimmunoprecipitating proteins, with Mr = 17,000‐100,000, were identified in silver‐stained gels. Western blots were then probed with antibodies to a series of candidate proteins that were chosen based on the molecular masses of the copurifying proteins. Two of these were identified as the molecular chaperones calnexin (90 kDa) and the immunoglobulin binding protein (BiP ; 78 kDa). Immunoprecipitation with antibodies to calnexin and BiP demonstrated that glycosylated AMPA receptor subunits were associated. The relationship between AMPA receptors and calnexin and BiP was further studied with immunocytochemistry of the hippocampus. Both calnexin and BiP labeling was present not only in the cell body but also in dendrites of hippocampal pyramidal neurons, where double‐label immunofluorescence also showed the presence of AMPA receptor subunits.
Effect of G Protein‐Coupled Receptor Kinase 2 on the Sensitivity of M4 Muscarinic Acetylcholine Receptors to Agonist‐Induced Internalization and Desensitization in NG108‐15 Cells
Authors: Holroyd E. W. , Szekeres P. G. , Whittaker R. D. , Kelly E. , Edwardson J. M.
Published: March 2003

Abstract : NG108‐15 cells express predominantly the M4 subtype of the muscarinic receptor for acetylcholine. Stimulation of these receptors by the agonist carbachol causes an inhibition of cellular adenylyl cyclase and a consequent fall in the intracellular cyclic AMP concentration. Pretreatment of the cells with carbachol caused both internalization and desensitization of the M4 receptor. Overexpression of G protein‐coupled receptor kinase (GRK) 2 caused an increase in the rate constant for receptor endocytosis (from 0.06 to 0.18 min‐1) and a decrease in the EC50 for carbachol stimulation of internalization (from 15 to 3 μM). Overexpression of a dominant negative form of GRK2 had more modest effects, reducing the rate constant for endocytosis (from 0.11 to 0.07 min‐1) and increasing the EC50 for carbachol stimulation of internalization (from 8 to 17 μM). Neither GRK2 nor dominant negative GRK2 overexpression had any effect on the rate constant for receptor recycling following agonist removal. The time course and extent of receptor desensitization in control cells were identical to the corresponding values for receptor internalization, and the rate and extent of desensitization were again increased by GRK2 overexpression. Exposure of the cells to hyperosmolar sucrose (0.6 M) almost completely blocked agonist‐induced receptor internalization in both control and GRK2‐overexpressing cells. Sucrose treatment also blocked agonist‐induced desensitization. We conclude that the internalization and desensitization of the M4 muscarinic receptor in NG108‐15 cells can be modulated in response to changes in GRK2 activity and also that internalization plays a key role in desensitization.
α2‐Macroglobulin Enhances the Clearance of Endogenous Soluble β‐Amyloid Peptide via Low‐Density Lipoprotein Receptor‐Related Protein in Cortical Neuron
Authors: Qiu Zhihua , Strickland Dudley K. , Hyman Bradley T. , Rebeck G. William
Published: March 2003

Abstract : Apolipoprotein E and α2‐macroglobulin

(α2M) are genetic risk factors for late‐onset Alzheimer's disease, and

both bind a cell surface receptor, the lowdensity lipoprotein receptor‐related

protein (LRP). To investigate the role of LRP on preventing the accumulation

of β‐amyloid peptide (Aβ), we examined the effects of α2M on

the clearance of endogenous Aβ. Studies were performed in primary Tg2576

transgenic mouse cortical neuronal cultures expressing human mutant amyloid

precursor protein (APP) 695. This system allowed us to follow endogenous

Aβ using immunoblots to detect monomeric forms of the peptide. Aβ

and APP levels were measured in conditioned media. We found that activated

α2M (α2M*) substantially decreased soluble Aβ

levels and had no effect on secreted or full‐length APP levels. Native

α2M, which is not a ligand for LRP, did not affect Aβ levels. The

receptor‐associated protein, which inhibits interaction of all ligands with

LRP in vitro, prevented α2M*‐induced decreases of soluble

Aβ levels. These data suggest that α2M* affects soluble

Aβ clearance rather than Aβ production. Further studies showed that

similar Aβ clearance via an LRP‐mediated pathway was observed after

treatment with another LRP ligand, lactoferrin. Taken together, these data

demonstrate that α2M* enhances the clearance of soluble Aβ via LRP in cortical neurons.
Botulinum Neurotoxin E‐Insensitive Mutants of SNAP‐25 Fail to Bind VAMP but Support Exocytosis
Authors: Washbourne Philip , Bortoletto Nicola , Graham Margaret E. , Wilson Michael C. , Burgoyne Robert D. , Montecucco Cesare
Published: March 2003

Abstract : Neurotransmitter release from synaptic vesicles is mediated by complex machinery, which includes the v‐and t‐SNAP receptors (SNAREs), vesicle‐associated membrane protein (VAMP), synaptotagmin, syntaxin, and synaptosome‐associated protein of 25 kDa (SNAP‐25). They are essential for neurotransmitter exocytosis because they are the proteolytic substrates of the clostridial neurotoxins tetanus neurotoxin and botulinum neurotoxins (BoNTs), which cause tetanus and botulism, respectively. Specifically, SNAP‐25 is cleaved by both BoNT/A and E at separate sites within the COOH‐terminus. We now demonstrate, using toxin‐insensitive mutants of SNAP‐25, that these two toxins differ in their specificity for the cleavage site. Following modification within the COOH‐terminus, the mutants completely resistant to BoNT/E do not bind VAMP but were still able to form a sodium dodecyl sulfate‐resistant complex with VAMP and syntaxin. Furthermore, these mutants retain function in vivo, conferring BoNT/E‐resistant exocytosis to transfected PC12 cells. These data provide information on structural requirements within the C‐terminal domain of SNAP‐25 for its function in exocytosis and raise doubts about the significance of in vitro binary interactions for the in vivo functions of synaptic protein complexes.
Characterization and Distribution of Ferritin Binding Sites in the Adult Mouse Brain
Authors: Hulet S. W. , Hess E. J. , Debinski W. , Arosio P. , Bruce K. , Powers S. , Connor J. R.
Published: March 2003

Abstract : Studies on iron uptake into the brain have traditionally
focused on transport by transferrin. However, transferrin receptors are not
found in all brain regions and are especially low in white matter tracts where
high iron concentrations have been reported. Several lines of research suggest
that a receptor for ferritin, the intracellular storage protein for iron, may
exist. We present, herein, evidence for ferritin binding sites in the brains
of adult mice. Autoradiographic studies using 125I‐recombinant
human ferritin demonstrate that ferritin binding sites in brain are
predominantly in white matter. Saturation binding analyses revealed a single
class of binding sites with a dissociation constant (KD)
of 4.65 × 10‐9M and a binding site density
(Bmax) of 17.9 fmol bound/μg of protein. Binding of radiolabeled ferritin can be competitively displaced by an excess of ferritin but not transferrin. Ferritin has previously been shown to affect cellular proliferation, protect cells from oxidative damage, and deliver iron. The significance of a cellular ferritin receptor is that ferritin is capable of delivering 2,000 times more iron per mole of protein than transferrin. The distribution of ferritin binding sites in brain vis‐à‐vis transferrin receptor distribution suggests distinct methods for iron delivery between gray and whi
Interleukin‐10 Inhibits Both Production of Cytokines and Expression of Cytokine Receptors in Microglia
Authors: Sawada Makoto , Suzumura Akio , Hosoya Hiromi , Marunouchi Tohru , Nagatsu Toshiharu
Published: March 2003

Abstract: Microglia, macrophage‐like cells in the CNS, are multifunctional cells; they play an important role in removal of dead cells or their remnants by phagocytosis in the CNS degeneration and are one of important cells in the CNS cytokine network to produce and respond to a variety of cytokines. The functions of microglia are regulated by inhibitory cytokines. We have reported the expression of interleukin (IL)‐10, one of the inhibitory cytokines, and its receptor in mouse microglia; therefore, IL‐10 may affect microglial functions. In this study, we investigated the effects of IL‐10 on purified microglia in culture. IL‐10 inhibited lipopolysaccharide‐induced IL‐1β and tumor necrosis factor‐α production, lysosomal enzyme activity, and superoxide anion production in a dose‐dependent manner, but did not affect granulocyte/macrophage colony‐stimulating factor‐dependent proliferation of microglia. IL‐10 also decreased the expression of both IL‐6 receptor and lipopolysaccharide‐induced IL‐2 receptor but not IL‐4 receptor on microglia as measured by flow cytometric analysis with an indirect immunofluorescence technique. IL‐10 also decreased mRNA expression of IL‐2 and IL‐6 cytokine receptors. These results suggest that IL‐10 is a unique and potent inhibitory factor in the CNS cytokine network involved in decreasing the expression of cytokine receptors as well as cytokine production by microglia.
Estrogen Modulates Neuronal Bcl‐xl Expression and β‐Amyloid‐Induced Apoptosis
Authors: Pike Christian J.
Published: March 2003

Abstract: Recent findings indicate that estrogen is neuroprotective, a cellular effect that may contribute to its clinical benefits in delaying the development of Alzheimer’s disease. In this report, we identify a novel neuronal action of estrogen that may contribute to its neuroprotective mechanism(s). Specifically, we report that estrogen significantly increases the expression of the antiapoptotic protin Bcl‐XL in cultured hippocampal neurons. This effect presumably reflects classic estrogen transcriptional regulation, as we identified a putative estrogen response element in the bcl‐X gene. Estrogen‐induced enhancement of Bcl‐XL is associated with a reduction in measures of β‐amyloid‐induced apoptosis, including inhibition of both caspase‐mediated proteolysis and neurotoxicity. A similar relationship between estrogen, Bcl‐XL expression, and resistance to degeneration was also observed in human hippocampus. We report neuronal colocalization of estrogen receptor and Bcl‐XL immunoreactivities that is most prominent in hippocampal subfield CA3, a region that shows relatively little immunoreactivity to paired helical filament‐1, a marker of Alzheimer’s disease neurodegeneration. These data suggest a novel mechanism of estrogen neuroprotection that may be relevant to estrogen’s suggested ability to modulate neuronal viability across the life span, from neural sexual differentiation and development through age‐related neurodegenerative conditions.
Second Messenger‐Regulated Protein Kinases in the Brain
Authors: Popoli Maurizio , Brunello Nicoletta , Perez Jorge , Racagni Giorgio
Published: March 2003

Abstract: Depression has been treated pharmacologically for over three decades, but the views regarding the mechanism of action of antidepressant drugs have registered recently a major change. It was increasingly appreciated that adaptive changes in postreceptor signaling pathways, rather than primary action of drugs on monoamine transporters, metabolic enzymes, and receptors, are connected to therapeutic effect. For some of the various signaling pathways affected by antidepressant treatment, it was shown that protein phosphorylation, which represents an obligate step for most pathways, is markedly affected by long‐term treatment. Changes were reported to be induced in the function of protein kinase C, cyclic AMP‐dependent protein kinase, and calcium/calmodulin‐dependent protein kinase. For two of these kinases (cyclic AMP‐ and calcium/calmodulin‐dependent), the changes have been studied in isolated neuronal compartments (microtubules and presynaptic terminals). Antidepressant treatment activates the two kinases and increases the endogenous phosphorylation of selected substrates (microtubule‐associated protein 2 and synaptotagmin). These modifications may be partly responsible for the changes induced by antidepressants in neurotransmission. The changes in protein phosphorylation induced by long‐term antidepressant treatment may contribute to explain the therapeutic action of antidepressants and suggest new strategies of pharmacological intervention.
Formation of the Base Modification 8‐Hydroxyl‐2′ ‐ Deoxyguanosine and DNA Fragmentation Following Seizures Induced by Systemic Kainic Acid in the Rat
Authors: Lan Jing , Henshall David C. , Simon Roger P. , Chen Jun
Published: March 2003

Abstract: The formation of oxidative DNA damage as a consequence of seizures remains little explored. We therefore investigated the regional and temporal profile of 8‐hydroxyl‐2′ ‐deoxyguanosine (8‐OHdG) formation, a hallmark of oxidative DNA damage and DNA fragmentation in rat brain following seizures induced by systemic kainic acid (KA). Formation of 8‐OHdG was determined via HPLC with electrochemical detection, and single‐ and double‐stranded DNA breaks were detected using in situ DNA polymerase I‐mediated biotin‐dATP nick‐translation (PANT) and terminal deoxynucleotidyl‐transferase‐mediated nick end‐labeling (TUNEL), respectively. Systemic KA (11 mg/kg) significantly increased levels of 8‐OHdG within the thalamus after 2 h, within the amygdala/piriform cortex after 4 h, and within the hippocampus after 8 h. Levels remained elevated up to sevenfold within these areas for 72 h. Smaller increases in 8‐OHdG levels were also detected within the parietal cortex and striatum. PANT‐positive cells were detected within the thalamus, amygdala/piriform cortex, and hippocampus 24‐72 h following KA injection. TUNEL‐positive cells appeared within the same brain regions and over a similar time course (24‐72 h) but were generally lower in number. The present data suggest oxidative damage to DNA may be an early consequence of epileptic seizures and a possible initiation event in the progression of seizure‐induced injury to DNA fragmentation and cell death.
Involvement of Mitogen‐Activated Protein Kinase in Agonist‐Induced Phosphorylation of the μ‐Opioid Receptor in HEK 293 Cells
Authors: Schmidt Harald , Schulz Stefan , Klutzny Marcus , Koch Thomas , Händel Manuela , Höllt Volker
Published: March 2003

Abstract: Agonist exposure of many G protein‐coupled receptors stimulates an activation of extracellular signal‐regulated protein kinases (ERKs) 1 and 2, members of the mitogen‐activated protein kinase (MAPK) family. Here, we show that treatment of human embryonic kidney (HEK) 293 cells stably transfected to express the rat μ‐opioid receptor (MOR1) with [D‐Ala2,MePhe4,Gly5‐ol]enkephalin (DAMGO) stimulated a rapid and transient (3‐5‐min) activation and nuclear translocation of MAPK. Exposure of these cells to the MAPK kinase 1 inhibitor PD98059 not only prevented MAPK activation but also inhibited homologous desensitization of the μ‐opioid receptor. We have therefore determined the effect of PD98059 on agonist‐induced μ‐receptor phosphorylation. DAMGO stimulated a threefold increase in MOR1 phosphorylation within 20 min that could be reversed by the antagonist naloxone. PD98059 produced a dose‐dependent inhibition of agonist‐promoted μ‐receptor phosphorylation with an IC50 of 20 μM. DAMGO also induced MOR1 internalization that peaked at 30 min. Confocal microscopy revealed that DAMGO‐induced MOR1 internalization was also largely inhibited in the presence of PD98059. U0126, another chemically unrelated inhibitor of the MAPK cascade, mimicked the effect of PD98059 on μ‐receptor phosphorylation and desensitization. MOR1 itself, however, appears to be a poor substrate for MAPK because μ‐receptors immunoprecipitated from stably transfected HEK 293 cells were not phosphorylated by exogenous ERK 2 in vitro. The fact that morphine also triggered MAPK activation but did not induce MOR1 internalization indicates that receptor internalization was not required for MOR1‐mediated mitogenic signaling. We conclude that MOR1 stimulates a rapid and internalization‐independent MAPK activation. Activation of the MAPK cascade in turn may not only relay mitogenic signals to the nucleus but also trigger initial events leading to phosphorylation and desensitization of the μ‐opioid receptor.
Amtyr1
Authors: Blenau Wolfgang , Balfanz Sabine , Baumann Arnd
Published: March 2003

Abstract: Biogenic amine receptors are involved in the regulation
and modulation of various physiological and behavioral processes in both
vertebrates and invertebrates. We have cloned a member of this gene family
from the CNS of the honeybee, Apis mellifera. The deduced amino acid
sequence is homologous to tyramine receptors cloned from Locusta
migratoria and Drosophila melanogaster as well as to an
octopamine receptor cloned from Heliothis virescens. Functional
properties of the honeybee receptor were studied in stably transfected human
embryonic kidney 293 cells. Tyramine reduced forskolin‐induced cyclic AMP
production in a dose‐dependent manner with an EC50 of ∼130
nM. A similar effect of tyramine was observed in membrane homogenates
of honeybee brains. Octopamine also reduced cyclic AMP production in the
transfected cell line but was both less potent (EC50 of ∼3
μM) and less efficacious than tyramine. Receptor‐encoding mRNA has a widespread distribution in the brain and subesophageal ganglion of the honeybee, suggesting that this tyramine receptor is involved in sensory signal processing as well as in higher‐order brain functions.
Apolipoprotein E Exhibits Isoform‐Specific Promotion of Lipid Efflux from Astrocytes and Neurons in Culture
Authors: Michikawa Makoto , Fan Qi‐Wen , Isobe Ichiro , Yanagisawa Katsuhiko
Published: March 2003

Abstract: Many studies have shown that apolipoprotein E (apoE) plays important roles in maintaining intracellular lipid homeostasis in nonneuronal cells. However, little is known about the extracellular transport of lipids in the CNS. In this study, we determined whether and to what degree lipid efflux from astrocytes and neurons depended on apoE. Our results showed that exogenously added apoE promoted the efflux of cholesterol and phosphatidylcholine from both astrocytes and neurons in culture, resulting in the generation of high‐density lipoprotein‐like particles. The order of potency of the apoE isoforms as lipid acceptors was apoE2 > apoE3 = apoE4 in astrocytes and apoE2 > apoE3 > apoE4 in neurons. Treatment with brefeldin A, monensin, and a protein kinase C inhibitor, H7, abolished the ability of apoE to promote cholesterol efflux from cultured astrocytes, without altering apoE‐mediated phosphatidylcholine efflux. In contrast, the efflux of both cholesterol and phosphatidylcholine promoted by apoE was abolished following treatment with heparinase or lactoferrin, which block the interaction of apoE with heparan sulfate proteoglycans (HSPGs) or low‐density lipoprotein receptor‐related protein (LRP), respectively. This study suggests that apoE promotes lipid efflux from astrocytes and neurons in an isoform‐specific manner and that cell surface HSPGs and/or HSPG‐LRP pathway may mediate this apoE‐promoted lipid efflux.
Cellular and Subcellular Localization of Peripheral Benzodiazepine Receptors After Trimethyltin Neurotoxicity
Authors: Kuhlmann Anthony C. , Guilarte Tomás R.
Published: March 2003

Abstract: The peripheral benzodiazepine receptor (PBR) is currently
used as a marker of inflammation and gliosis following brain injury. Previous
reports suggest that elevated PBR levels in injured brain tissue are specific
to activated microglia and infiltrating macrophages. We have produced
hippocampal lesions using the neurotoxicant trimethyltin (TMT) to examine the
cellular and subcellular nature of the PBR response. Degenerating,
argyrophilic pyramidal neurons were observed in the hippocampus at 2 and 14
days after TMT exposure. Reactive microglia were also evident at both times
with a maximal response observed at 14 days, subsiding by 6 weeks.
Astrocytosis was observed at 14 days and 6 weeks, but not 2 days, after TMT
administration, suggesting that the onset of the astroglia response is
delayed, but more persistent, compared with microgliosis. Morphological
evidence from [3H]PK11195 microautoradiography and PBR
immunohistochemistry indicates that both astrocytes and microglia are capable
of expressing high levels of PBR after injury. This was confirmed by double
labeling of either Griffonia simplicifolia isolectin B4, a microglial‐specific marker, or glial fibrillary acidic protein, an astrocyte‐specific protein with PBR fluorescence immunohistochemistry. These results demonstrate that PBR expression is increased after brain injury in both activated microglia and astrocytes. Our findings also provide the first evidence for in situ nuclear localization of PBR in glial cells.
Identification of Variants and Dual Promoters of Murine Serine/Threonine Kinase KKIAMRE
Authors: Sassa Takayuki , Gomi Hiroshi , Sun William , Ikeda Toshio , Thompson Richard F. , Itohara Shigeyoshi
Published: March 2003

Abstract: KKIAMRE is a serine/threonine protein kinase whose transcripts increase in the deep cerebellar nuclei of the rabbit after eyeblink conditioning, a model of associative learning and memory. We here characterized the expression, isoforms, and promoters of murine KKIAMRE gene. The expression of KKIAMRE was detected, by in situ hybridization and immunohistochemistry, in neurons in various brain regions including deep cerebellar nuclei. The gene spans ∼40 kb and consists of 15 exons. Analysis of cDNA clones revealed multiple variants, having diversity in the putative carboxy‐terminal regulatory domain, generated by alternative splicing and intraexonal termination. Furthermore, they had alternative 5′ noncoding sequences. Primer extension, RNase protection, and transient expression assays revealed that two alternative promoters linked to distinct noncoding exons direct the expression of KKIAMRE. The gene was mapped on chromosomes 5 and 4 in mouse and human, respectively.
Dopamine‐Induced Apoptosis Is Mediated by Oxidative Stress and Is Enhanced by Cyanide in Differentiated PC12 Cells
Authors: Jones Douglas C. , Gunasekar Palur G. , Borowitz Joseph L. , Isom Gary E.
Published: March 2003

Abstract: Dopamine (DA) oxidation and the generation of reactive oxygen species (ROS) may contribute to the degeneration of dopaminergic neurons underlying various neurological conditions. The present study demonstrates that DA‐induced cytotoxicity in differentiated PC12 cells is mediated by ROS and mitochondrial inhibition. Because cyanide induces parkinson‐like symptoms and is an inhibitor of the antioxidant system and mitochondrial function, cells were treated with KCN to study DA toxicity in an impaired neuronal system. Differentiated PC12 cells were exposed to DA, KCN, or a combination of the two for 12‐36 h. Lactate dehydrogenase (LDH) assays indicated that both DA (100‐500 μM) and KCN (100‐500 μM) induced a concentration‐ and time‐dependent cell death and that their combination produced an increase in cytotoxicity. Apoptotic death, measured by Hoechst dye and TUNEL (terminal deoxynucleotidyltransferase dUTP nick end‐labeling) staining, was also concentration‐ and time‐dependent for DA and KCN. DA plus KCN produced an increase in apoptosis, indicating that KCN, and thus an impaired system, enhances DA‐induced apoptosis. To study the mechanism(s) of DA toxicity, cells were pretreated with a series of compounds and incubated with DA (300 μM) and/or KCN (100 μM) for 24 h. Nomifensine, a DA reuptake inhibitor, rescued nearly 60‐70% of the cells from DA‐ and DA plus KCN‐induced apoptosis, suggesting that DA toxicity is in part mediated intracellularly. Pretreatment with antioxidants attenuated DA‐ and KCN‐induced apoptosis, indicating the involvement of oxidative species. Furthermore, buthionine sulfoximine, an inhibitor of glutathione synthesis, increased the apoptotic response, which was reversed when cells were pretreated with antioxidants. DA and DA plus KCN produced a significant increase in intracellular oxidant generation, supporting the involvement of oxidative stress in DA‐induced apoptosis. The nitric oxide synthase inhibitor NG‐nitro‐L‐arginine methyl ester and the peroxynitrite scavenger uric acid blocked apoptosis and oxidant production, indicating involvement of nitric oxide. These results suggest that DA neurotoxicity is enhanced under the conditions induced by cyanide and involves both ROS and nitric oxide‐mediated oxidative stress as an initiator of apoptosis.
A2A Adenosine Receptor Facilitation of Neuromuscular Transmission
Authors: Correia‐de‐Sá P. , Timóteo M. A. , Ribeiro J. A.
Published: March 2003

Abstract: The influence of stimulus pulse duration on calcium mobilization triggering facilitation of evoked [3H]acetylcholine ([3H]ACh) release by the A2A adenosine receptor agonist CGS 21680C was studied in the rat phrenic nerve‐hemidiaphragm. The P‐type calcium channel blocker ω‐agatoxin IVA (100 nM) decreased [3H]ACh release evoked with pulses of 0.04‐ms duration, whereas nifedipine (1 μM) inhibited transmitter release with pulses of 1‐ms duration. Depletion of intracellular calcium stores by thapsigargin (2 μM) decreased [3H]ACh release evoked by pulses of 1 ms, an effect observed even in the absence of extracellular calcium. With short (0.04‐ms) stimulation pulses, when P‐type calcium influx triggered transmitter release, facilitation of [3H]ACh release by CGS 21680C (3 nM) was attenuated by both thapsigargin (2 μM) and nifedipine (1 μM). With longer stimuli (1 ms), a situation in which both thapsigargin‐sensitive internal stores and L‐type channels are involved in ACh release, pretreatment with either ω‐agatoxin IVA (100 nM) or nifedipine (1 μM) reduced the facilitatory effect of CGS 21680C (3 nM). The results suggest that A2A receptor activation facilitates ACh release from motor nerve endings through alternatively mobilizing the available calcium pools (thapsigargin‐sensitive internal stores and/or P‐ or L‐type channels) that are not committed to the release process in each stimulation condition.
Nerve Growth Factor‐Induced Differentiation Does Not Alter the Biochemical Properties of a Mutant Prion Protein Expressed in PC12 Cells
Authors: Chiesa Roberto , Harris David A.
Published: March 2003

Abstract : Insertional and point mutations in the gene encoding the

prion protein (PrP) are responsible for familial prion diseases. We have

previously generated lines of Chinese hamster ovary cells that express PrP

molecules carrying pathogenic mutations, and found that the mutant proteins

display several biochemical properties reminiscent of PrPSc, the

infectious isoform of PrP. To analyze the properties and effects of mutant PrP

molecules expressed in cells with a neuronal phenotype, we have constructed

stably transfected lines of PC12 cells that synthesize a PrP molecule carrying

a nine‐octapeptide insertion. We report here that this mutant PrP acquires

scrapie‐like properties, including detergent insolubility, protease

resistance, and resistance to phospholipase cleavage of its glycolipid anchor.

A detergent‐insoluble and phospholipase‐resistant form of the mutant protein

is also released spontaneously into conditioned medium. These scrapie‐like

biochemical properties are quantitatively similar to those seen in Chinese

hamster ovary cells and are not affected by differentiation of the PC12 cells

into sympathetic neurons by nerve growth factor. Moreover, there is no

detectable effect of mutant PrP expression on the morphology or viability of

the cells in either the differentiated or undifferentiated state. These

results indicate that conversion of mutant PrP into a PrPSc‐like form does not depend critically on the cellular context, and they suggest that mutant PrP expressed in cultured cells, even those having the phenotype of differentiated neurons, is not neurotoxic.
Neurons Overexpressing Heme Oxygenase‐1 Resist Oxidative Stress‐Mediated Cell Death
Authors: Chen Kai , Gunter Karlene , Maines Mahin D.
Published: March 2003

Abstract : This is the first report on the protective effect of heme

oxygenase‐1 (HO‐1) overexpression against oxidative stress‐mediated neuronal

cell death and demonstration of a decreased production of oxygen free radicals

when HO‐1 levels are increased. HO‐1 is the heat shock/stress cognate of the

heat shock protein 32 family of proteins. A known function of these proteins

is α‐meso bridge‐specific cleavage of the heme molecule. For the present

study, we used cerebellar granular neurons (CGNs) isolated from homozygous

transgenic (Tg) mice that overexpress HO‐1 under neuron‐specific enolase

control and nontransgenic (Ntg) littermates. The Tg mouse CGNs were

characterized by increased levels of HO‐1 mRNA and protein, a lower resting

intracellular calcium concentration, and a reduced HO‐1 transcriptional

response to glutamate‐mediated oxidative stress. Compared with the Ntg

neurons, when exposed to glutamate (30 μM or 3 mM), the

magnitude of cell viability was increased and the number of cells exhibiting

membrane permeability and chromatin condensation were significantly decreased

in the Tg CGN cultures. The population of neurons surviving glutamate toxicity

decreased when HO‐1 activity was inhibited by a peptide inhibitor. The

neuroprotective effect by HO‐1 was extended to

H2O2‐induced cell death. The mechanism of protection may involve in part a reduced production of reactive oxygen species upon exposure to glutamate. We suggest that induction of HO‐1 by pharmacological means may be a novel approach to amelioration of oxidative insults to neurons.
Gα12 and Gα13 Inhibit Ca2+ ‐Dependent Exocytosis Through Rho/Rho‐Associated Kinase‐Dependent Pathway
Authors: Yamaguchi Yoshiaki , Katoh Hironori , Yasui Hidekazu , Aoki Junko , Nakamura Kazuhiro , Negishi Manabu
Published: March 2003

The release of neurotransmitters is known to be regulated by activation of heterotrimeric G protein‐coupled receptors, although precise mechanisms have not yet been elucidated. To assess the role of the G12 family of

heterotrimeric G proteins in the regulation of neurotransmitter release, we

established PC12 cell lines that expressed constitutively active

Gα12 or Gα13 using an

isopropyl‐β‐d‐thiogalactoside‐inducible expression system. In

the cells, expression of constitutively active Gα12 or

Gα13 inhibited the high K+ ‐evoked

[3H]dopamine release without any effect on the high

K+‐induced increase in intracellular Ca2+ concentration.

A Ca2+ ionophore ionomycin‐induced [3H]dopamine release

was also inhibited by the expression of active Gα12 or

Gα13. These inhibitory effects of Gα12 and

Gα13 on [3H]dopamine release were mimicked by the

expression of constitutively active RhoA. In addition, Y‐27632, and inhibitor

of Rho‐associated kinase, a downstream Rho effector, completely abolished the

inhibition of [3H]dopamine release by Gα12,

Gα13, and RhoA. These results indicate that Ca2+

‐dependent exocytosis is regulated by Gα12 and

Gα13 through a Rho/Rho‐associated kinase‐dependent pathway.
Benzodiazepine‐Sensitive GABAA Receptors Limit the Activity of the NMDA/NO/Cyclic GMP Pathway
Authors: Fedele Ernesto , Ansaldo Maria Antonia , Varnier Giorgia , Raiteri Maurizio
Published: March 2003

In the cerebellum, infusion of NMDA (200 μM) for 20 min evoked

a marked (200%) increase of extracellular cyclic GMP (cGMP) levels. The

selective GABAA receptor agonist muscimol (0.01‐100 μM)

was able to counteract the NMDA effect with an EC50 of 0.65

μM ; the inhibitory effect of muscimol (10 μM) was

prevented by bicuculline (50 μM). Diazepam (10 μM)

significantly potentiated the muscimol (1 μM) inhibition ;

furthermore, when coinfused with 0.1 μM muscimol (a concentration

not affecting, on its own, the cGMP response to NMDA), diazepam (10

μM) reduced the NMDA effect. Similar results were obtained with

zolpidem (0.1‐1 μM). Finally, local infusion of the benzodiazepine

site antagonist flumazenil (10 μM), together with muscimol and

diazepam, almost completely restored the effect of NMDA on extracellular cGMP

levels. It is concluded that GABAA receptors potently control the

NMDA/nitric oxide/cGMP pathway in the cerebellum in vivo. In terms of the

α subunit composition, we can deduce that the cerebellar

GABAA receptor does not contain α6 or

β4 subunits because it is diazepam‐sensitive. Moreover, the

observation that zolpidem is active at a rather low concentration, in

combination with localization studies present in the literature, tend to

exclude the presence of α5 subunits in the receptor

composition and suggest the involvement of an α1 subunit.
Malonate and 3‐Nitropropionic Acid Neurotoxicity Are Reduced in Transgenic Mice Expressing a Caspase‐1 Dominant‐Negative Mutant
Authors: Andreassen Ole A , Ferrante Robert J , Hughes Duncan B , Klivenyi Peter , Dedeoglu Alpaslan , Ona Victor O , Friedlander Robert M , Beal M Flint
Published: March 2003

Increasing evidence implicates caspase‐1‐mediated cell death as a major mechanism of neuronal death in neurodegenerative diseases. In the present study we investigated the role of caspase‐1 in neurotoxic experimental animal models of Huntington's disease (HD) by examining whether transgenic mice expressing a caspase‐1 dominant‐negative mutant are resistant to malonate and 3‐nitropropionic acid (3‐NP) neurotoxicity. Intrastriatal injection of malonate resulted in significantly smaller striatal lesions in mutant caspase‐1 mice than those observed in littermate control mice. Caspase‐1 was significantly activated following malonate intrastriatal administration in control mice but significantly attenuated in mutant caspase‐1 mice. Systemic 3‐NP treatment induced selective striatal lesions that were significantly smaller within mutant caspase‐1 mice than in littermate control mice. These results provide further evidence of a functional role for caspase‐1 in both malonate‐ and 3‐NP‐mediated neurotoxin models of HD.
Mitochondrial Dysfunction in Cybrid Lines Expressing Mitochondrial Genes from Patients with Progressive Supranuclear Palsy
Authors: Swerdlow R. H. , Golbe L. I. , Parks J. K. , Cassarino D. S. , Binder D. R. , Grawey A. E. , Litvan I. , Bennett J. P. , Wooten G. F. , Parker W. D.
Published: March 2003

Abstract: Progressive supranuclear palsy (PSP) is a
neurodegenerative movement disorder of unknown etiology. We hypothesized that
mitochondrial DNA (mtDNA) aberration could occur in this disease and
contribute to its pathogenesis. To address this we created transmitochondrial
cytoplasmic hybrid (cybrid) cell lines expressing mitochondrial genes from
persons with PSP. The presence of cybrid mtDNA aberration was screened for by
biochemical assay of mitochondrial gene products. Relative to a control cybrid
set, complex I activity was reduced in PSP cybrid lines (p < 0.005). Antioxidant enzyme activities were elevated in PSP cybrid lines. These data suggest that mtDNA aberration occurs in PSP, causes electron transport chain pathology, and can produce oxidative stress. Further study of mitochondrial dysfunction in PSP may yield insights into why neurodegeneration occurs in this disease.
Induction of Oxidative DNA Damage in the Peri‐Infarct Region After Permanent Focal Cerebral Ischemia
Authors: Nagayama Tetsuya , Lan Jing , Henshall David C. , Chen Dexi , O'Horo Cristine , Simon Roger P. , Chen Jun
Published: March 2003

Abstract: To address the role of oxidative DNA damage in focal cerebral ischemia lacking reperfusion, we investigated DNA base and strand damage in a rat model of permanent middle cerebral artery occlusion (MCAO). Contents of 8‐hydroxyl‐2′‐deoxyguanosine (8‐OHdG) and apurinic/apyrimidinic abasic sites (AP sites), hallmarks of oxidative DNA damage, were quantitatively measured in nuclear DNA extracts from brains obtained 4‐72 h after MCAO. DNA single‐ and double‐strand breaks were detected on coronal brain sections using in situ DNA polymerase I‐mediated biotin‐dATP nick‐translation (PANT) and terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling (TUNEL), respectively. Levels of 8‐OHdG and AP sites were markedly elevated 16‐72 h following MCAO in the frontal cortex, representing the peri‐infarct region, but levels did not significantly change within the ischemic core regions of the caudateputamen and parietal cortex. PANT‐ and TUNEL‐positive cells began to be detectable 4‐8 h following MCAO in the caudate‐putamen and parietal cortex and reached maximal levels at 72 h. PANT‐ and TUNEL‐positive cells were also detected 16‐72 h after MCAO in the lateral frontal cortex within the infarct border, where many cells also showed colocalization of DNA single‐strand breaks and DNA fragmentation. In contrast, levels of PANT‐positive cells alone were transiently increased (16 h after MCAO) in the medial frontal cortex, an area distant from the infarct zone. These data suggest that within peri‐infarct brain regions, oxidative injury to nuclear DNA in the form of base and strand damage may be a significant and contributory cause of secondary expansion of brain damage following permanent focal ischemia.
The Cellular Prion Protein Colocalizes with the Dystroglycan Complex in the Brain
Authors: Keshet Gilmor I. , Bar‐Peled Osnat , Yaffe David , Nudel Uri , Gabizon Ruth
Published: March 2003

Abstract: The function of PrPC, the cellular prion
protein (PrP), is still unknown. Like other glycophosphatidylinositol‐anchored
proteins, PrP resides on Triton‐insoluble, cholesterol‐rich membranous
microdomains, termed rafts. We have recently shown that the activity and
subcellular localization of the neuronal isoform of nitric oxide synthase
(nNOS) are impaired in adult PrP0/0 mice as well as in scrapie‐infected mice. In this study, we sought to determine whether PrP and nNOS are part of the same functional complex and, if so, to identify additional components of such a complex. To this aim, we looked for proteins that coimmunoprecipitated with PrP in the presence of detergents either that completely dissociate rafts, to identify stronger interactions, or that preserve the raft structure, to identify weaker interactions. Using this detergent‐dependent immunoprecipitation protocol we found that PrP interacts strongly with dystroglycan, a transmembrane protein that is the core of the dystrophin—glycoprotein complex (DGC). Additional results suggest that PrP also interacts with additional members of the DGC, including nNOS. PrP coprecipitated only with established presynaptic proteins, consistent with recent findings suggesting that PrP is a presynaptic protein.
Nitric Oxide‐Sensitive Guanylyl Cyclase Activity Inhibition Through Cyclic GMP‐Dependent Dephosphorylation
Authors: Ferrero Rut , Rodríguez‐Pascual Fernando , Miras‐Portugal M. Teresa , Torres Magdalena
Published: March 2003

Abstract: The soluble form of guanylyl cyclase (sGC) plays a pivotal role in the transduction of inter‐ and intracellular signals conveyed by nitric oxide. Here, a feedback inhibitory mechanism triggered by cyclic guanosine‐3′,5′‐monophosphate (cGMP)‐dependent protein kinase (PKG) activation is described. Preincubation of chromaffin cells with C‐type natriuretic peptide, which increased cGMP levels and activated PKG, or with cGMP‐permeant analogue (which also activates PKG), in the presence of a broad‐spectrum phosphodiesterase inhibitor, resulted in a decrease in subsequent sodium nitroprusside (SNP)‐dependent cGMP elevations. This inhibitory effect was mimicked by activating a protein phosphatase and counteracted by the selective PKG inhibitor KT‐5823 and by different protein phosphatase inhibitors. Immunoprecipitation of sGC from cells submitted to different treatments followed by immunodetection with antiphosphoserine antibodies (clone 4A9) showed changes in phosphorylation levels of the β subunit of sGC, and these changes correlated well with differences in SNP‐elicited cGMP accumulations. Pretreatment of cells with several PKG inhibitors or protein phosphatase inhibitors produced an enhancement of SNP‐stimulated cGMP rises without changing the SNP concentration required to produce half‐maximal or maximal responses. Taken together, these results indicate that the catalytic activity of sGC is closely coupled to the phosphorylation state of its β subunit and that the tonic activity of PKG or its stimulation regulates sGC activity through dephosphorylation of the β subunit.
Lipid Alterations in Transient Forebrain Ischemia
Authors: Rao A. Muralikrishna , Hatcher J. F. , Dempsey R. J.
Published: March 2003

Abstract: We have previously demonstrated that cytidine5′‐diphosphocholine (CDP‐choline or citicoline) attenuated arachidonicacid (ArAc) release and provided significant protection for the vulnerablehippocampal CA1 neurons of the cornu ammonis after transientforebrain ischemia of gerbil. ArAc is released by the activation ofphospholipases and the alteration of phosphatidylcholine (PtdCho) synthesis.Released ArAc is metabolized by cyclooxygenases/lipoxygenases to formeicosanoids and reactive oxygen species (ROS). ROS contribute to neurotoxicitythrough generation of lipid peroxides and the cytotoxic byproducts4‐hydroxynonenal and acrolein. ArAc can also stimulate sphingomyelinase toproduce ceramide, a potent pro‐apoptotic agent. In the present study, weexamined the changes and effect of CDP‐choline on ceramide and phospholipidsincluding PtdCho, phosphatidylethanolamine (PtdEtn), phosphatidylinositol(PtdIns), phosphatidylserine (PtdSer), sphingomyelin, and cardiolipin (anexclusive inner mitochondrial membrane lipid essential for electron transport)following ischemia/1‐day reperfusion. Our studies indicated significantdecreases in total PtdCho, PtdIns, PtdSer, sphingomyelin, and cardiolipin andloss of ArAc from PtdEtn in gerbil hippocampus after 10‐min forebrainischemia/1‐day reperfusion. CDP‐choline (500 mg/kg i.p. immediately afterischemia and at 3‐h reperfusion) significantly restored the PtdCho,sphingomyelin, and cardiolipin levels as well as the ArAc content of PtdChoand PtdEtn but did not affect PtdIns and PtdSer. These data suggest multiplebeneficial effects of CDP‐choline: (1) stabilizing the cell membrane byrestoring PtdCho and sphingomyelin (prominent components of outer cellmembrane), (2) attenuating the release of ArAc and limiting its oxidativemetabolism, and (3) restoring cardiolipin levels.