bluuebunny/crossref_metadata_2025 huggingface dataset:
Charge Alterations of E22 Enhance the Pathogenic Properties of the Amyloid β‐Protein
Authors: Melchor Jerry P. , McVoy Lauren , Van Nostrand William E.
Published: March 2003

Abstract: Cerebral amyloid angiopathy (CAA) due to amyloid β‐protein (Aβ) is a key pathological feature of patients with Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis, Dutch‐type (HCHWA‐D). The CAA in these disorders is characterized by deposition of Aβ in the smooth muscle cells within the cerebral vessel wall. Recently, a new mutation in Aβ, E22K, was identified in several Italian families that, like HCHWA‐D, is associated with CAA and hemorrhagic stroke. These two similar disorders, stemming from amino acid substitutions at position 22 of Aβ, implicate the importance of this site in the pathology of HCHWA. Previously we showed that HCHWA‐D Aβ1‐40 containing the E22Q substitution induces robust pathologic responses in cultured human cerebrovascular smooth muscle cells (HCSM cells), including highly elevated levels of cell‐associated Aβ precursor (AβPP) and cell death. In the present study, a series of E22 mutant Aβ1‐40 peptides were synthesized, and their pathogenic properties toward cultured HCSM cells were evaluated. Quantitative fluorescence analyses showed that mutant Aβ1‐40 peptides either containing a loss of charge (E22Q and E22A) or a change of charge (E22K) bind to the surface of HCSM cells and form amyloid fibrils. Similarly, this same group of E22 mutant Aβ1‐40 peptides caused enhanced pathologic responses in HCSM cells. In contrast, wild‐type E22 or the charge‐preserving E22D Aβ1‐40 peptides were devoid of any of these pathogenic properties. These data suggest that a change or loss of charge at position 22 of Aβ enhances the pathogenic effects of the peptide toward HCSM cells and may contribute to the pathogenesis of the phenotypically related HCHWA disorders.
The Ginkgo biloba Extract (EGb 761) Protects and Rescues Hippocampal Cells Against Nitric Oxide‐Induced Toxicity
Authors: Bastianetto Stéphane , Zheng Wen‐Hua , Quirion Rémi
Published: March 2003

Abstract: An excess of the free radical nitric oxide (NO) is viewed as a deleterious factor involved in various CNS disorders. Numerous studies have shown that the Ginkgo biloba extract EGb 761 is a NO scavenger with neuroprotective properties. However, the mechanisms underlying its neuroprotective ability remain to be fully established. Thus, we investigated the effect of different constituents of EGb 761, i.e., flavonoids and terpenoids, against toxicity induced by NO generators on cells of the hippocampus, a brain area particularly susceptible to neurodegenerative damage. Exposure of rat primary mixed hippocampal cell cultures to either sodium nitroprusside (SNP; 100 μM) or 3‐morpholinosydnonimine resulted in both a decrease in cell survival and an increase in free radical accumulation. These SNP‐induced events were blocked by either EGb 761 (10‐100 μg/ml) or its flavonoid fraction CP 205 (25 μg/ml), as well as by inhibitors of protein kinase C (PKC; chelerythrine) and L‐type calcium channels (nitrendipine). In contrast, the terpenoid constituents of EGb 761, known as bilobalide and ginkgolide B, as well as inhibitors of phospholipases A [3‐[(4‐octadecyl)benzoyl]acrylic acid (OBAA)] and C (U‐73122), failed to display any significant effects. Moreover, EGb 761 (50 μg/ml), CP 205 (25 μg/ml), and chelerythrine were also able to rescue hippocampal cells preexposed to SNP (up to 1 mM). Finally, EGb 761 (100 μg/ml) was shown to block the activation of PKC induced by SNP (100 μM). These data suggest that the protective and rescuing abilities of EGb 761 are not only attributable to the antioxidant properties of its flavonoid constituents but also via their ability to inhibit NO‐stimulated PKC activity.
gp120 Induces Cell Death in Human Neuroblastoma Cells Through the CXCR4 and CCR5 Chemokine Receptors
Authors: Catani M. Valeria , Corasaniti M. Tiziana , Navarra Michele , Nisticò Giuseppe , Finazzi‐Agrò Alessandro , Melino Gerry
Published: March 2003

Abstract: To infect target cells, the human immunodeficiency virus (HIV) type I (HIV‐1) must engage not only the well‐known CD4 molecule, but it also requires one of several recently described coreceptors. In particular, the CXCR4 (LESTR/fusin) receptor allows fusion and entry of T‐tropic strains of HIV, whereas CCR5 is the major coreceptor used by primary HIV‐1 strains that infect macrophages and CD4+ T‐helper cells (M‐tropic viruses). In addition, the α chemokine SDF1α and the β chemokines MIP1α, MIP1β, and RANTES, natural ligands of CXCR4 and CCR5, respectively, are potent soluble inhibitors of HIV infection by blocking the binding between the viral envelope glycoprotein gp120 and the coreceptors. Approximately two‐thirds of individuals with acquired immunodeficiency syndrome (AIDS) show neurologic complications, which are referred to a syndrome called AIDS dementia complex or HIV‐1‐associated cognitive/motor complex. The HIV‐1 coat glycoprotein gp120 has been proposed as the major etiologic agent for neuronal damage, mediating both direct and indirect effects on the CNS. Furthermore, recent findings showing the presence of chemokine receptors on the surface of different cell types resident in the CNS raise the possibility that the association of gp120 with these receptors may contribute to the pathogenesis of neurological dysfunction. Here, we address the possible role of α and β chemokines in inhibiting gp120‐mediated neurotoxicity using the human neuroblastoma CHP100 cell line as an experimental model. We have previously shown that, in CHP100 cells, picomolar concentrations of gp120 produce a significant increase in cell death, which seems to proceed through a Ca2+ ‐ and NMDA receptor‐dependent cascade. In this study, we gained insight into the mechanism(s) of neurotoxicity elicited by the viral glycoprotein. We found that CHP100 cells constitutively express both CXCR4 and CCR5 receptors and that stimulation with phorbol 12‐myristate 13‐acetate down‐regulates their expression, thus preventing gp120‐induced cell death. Furthermore, all the natural ligands of these receptors exerted protective effects against gp120‐mediated neuronal damage, although with different efficiencies. These findings, together with our previous reports, suggest that the neuronal injury observed in HIV‐1 infection could be due to direct (or indirect) interactions between the viral protein gp120 and chemokine and/or NMDA receptors.
GABAA‐Mediated Toxicity of Hippocampal Neurons In Vitro
Authors: Lukasiuk Katarzyna , Pitkänen Asla
Published: March 2003

Abstract: In the present study, we examined whether the elevation of GABA by γ‐vinyl‐GABA protects cultured rat fetal hippocampal neurons against toxicity induced by a 20‐min incubation with 100 μM L‐glutamate. Neither a 24‐h pretreatment nor posttreatment with γ‐vinyl‐GABA (100 μM) had any neuroprotective effects, as determined by counting microtubule‐associated protein‐2 positive cells and lactate dehydrogenase assay 24 h after the glutamate treatment. Unexpectedly, γ‐vinyl‐GABA alone induced a 20% loss of microtubule‐associated protein‐2‐positive cells in a culture that was grown in medium containing 25 mM KCl. The toxic effect of γ‐vinyl‐GABA was mimicked by a 24‐h treatment with GABA (100 μM) and the GABAA receptor agonist, muscimol (10 μM), but not the GABAB receptor agonist, baclofen (10 μM). The GABAA receptor antagonist, bicuculline (10 μM), protected against γ‐vinyl‐GABA and GABA‐evoked toxicity. Neither γ‐vinyl‐GABA nor GABA was toxic in culture medium containing 15 mM KCl. These data indicate that, under depolarizing conditions, an increased GABA level is toxic for a subpopulation of developing hippocampal neurons in vitro. The effect is GABAA receptor‐mediated. These data provide a new view for understanding neurodegenerative processes, and raise a question of the safety of therapies aimed at increasing GABA concentration following brain insults, especially in immature brains.
Neuronal and Extraneuronal Expression and Regulation of the Human α5 Nicotinic Receptor Subunit Gene
Authors: Flora Adriano , Schulz Regine , Benfante Roberta , Battaglioli Elena , Terzano Susanna , Clementi Francesco , Fornasari Diego
Published: March 2003

Abstract : The mRNA encoding the human α5 nicotinic subunit was detected in several structures of the nervous system but appeared to be mainly expressed in cerebellum, thalamus, and the autonomic ganglia. For the first time, the α5 transcript was also detected in several non‐neuronal tissues, with maximal expressions being found throughout the gastrointestinal tract, thymus, and testis. Many other extraneuronal sites expressed α5, but there were also nonexpressing organs, such as the liver, spleen, and kidney. To understand the transcriptional mechanisms controlling such a diversified expression of α5 in neuronal and nonneuronal cells, we isolated the 5′‐regulatory region of the human gene and characterized its properties. Here we identify the α5 core promoter and demonstrate that the DNA regions surrounding it contain elements (with positive or negative activities) that work in a tissue‐specific fashion. In particular, the segment specifying the 5′‐untranslated region in neuronal cells has most of the properties of an enhancer because it activates a heterologous promoter in a position‐ and orientation‐independent fashion. We therefore conclude that the expression of α5 relies on a highly complex promoter that uses distinct regulatory elements to comply with the different functional and developmental requirements of the various tissues and organs.
N‐Acetylaspartylglutamate
Authors: Neale Joseph H , Bzdega Tomasz , Wroblewska Barbara
Published: March 2003

In the progress of science, as in life, timing is important. The acidic dipeptide, N‐acetylaspartylglutamate (NAAG), was discovered in the

mammalian nervous system in 1965, but initially was not considered to be a

neurotransmitter candidate. In the mid‐1980s, a few laboratories revisited the

question of NAAG's role in the nervous system and pursued hypotheses regarding

its function that ranged from a precursor for the transmitter pool of

glutamate to a direct role as a peptide transmitter. Since that time, NAAG has

been tested against nearly all of the established criteria for identification

of a neurotransmitter. It successfully meets each of these tests, including a

concentrated presence in neurons and synaptic vesicles, release from axon

endings in a calcium‐dependent manner following initiation of action

potentials, and extracellular hydrolysis by membrane‐bound peptidase activity.

NAAG is the most prevalent and widely distributed neuropeptide in the

mammalian nervous system. NAAG activates NMDA receptors with a low potency

that may vary among receptor subtypes, and it is a highly selective agonist at

the type 3 metabotropic glutamate receptor (mGluR3). Acting through this

receptor, NAAG reduces cyclic AMP levels, decreases voltage‐dependent calcium

conductance, suppresses excitotoxicity, influences long‐term potentiation and

depression, regulates GABAA receptor subunit expression, and inhibits synaptic release of GABA from cortical neurons. Cloning of peptidase activities against NAAG provides opportunities to study the cellular and molecular mechanisms by which synaptic NAAG peptidase activity is controlled. Given the codistribution of this peptide with a spectrum of traditional transmitters and its ability to activate mGluR3, we speculate that one role for NAAG following synaptic release is the activation of metabotropic autoreceptors that inhibit subsequent transmitter release. A second role is the production of extracellular glutamate following NAAG hydrolysis.
Functions of N‐Acetyl‐l‐Aspartate and N‐Acetyl‐l‐Aspartylglutamate in the Vertebrate Brain
Authors: Baslow Morris H
Published: March 2003

N‐Acetyl‐l‐aspartate (NAA) and its derivative N‐acetylaspartylglutamate (NAAG) are major osmolytes present in the vertebrate brain. Although they are synthesized primarily in neurons, their function in these cells is unclear. In the brain, these substances undergo intercompartmental cycles in which they are released by neurons in a regulated fashion and are then rapidly hydrolyzed by catabolic enzymes associated with glial cells. Recently, the catabolic enzyme for NAA hydrolysis has been found to be expressed only in oligodendrocytes, and the catabolic enzyme for NAAG expressed only in astrocytes. These results indicate an unusual tricellular metabolic sequence for the synthesis and hydrolysis of NAAG wherein it is synthesized in neurons from NAA and l‐glutamate, hydrolyzed to NAA and l‐glutamate by astrocytes, and further hydrolyzed to l‐aspartate and acetate by oligodendrocytes. Since the discovery that the NAA and NAAG anabolic products of neurons are specifically targeted to oligodendrocytes and astrocytes, respectively, this unique metabolic compartmentalization also suggests that these substances may play an important role in cell‐specific glial signaling. In this review, it is hypothesized that a key function of NAA and NAAG in the vertebrate brain is in cell signaling and that these substances are important in the regulation of interactions of brain cells and in the establishment and maintenance of the nervous system.
A Possible Role of Alanine for Ammonia Transfer Between Astrocytes and Glutamatergic Neurons
Authors: Waagepetersen Helle S , Sonnewald Ursula , Larsson Orla M , Schousboe Arne
Published: March 2003

The metabolism of [U‐13C]lactate (1 mM) in the presence of unlabeled glucose (2.5 mM) was investigated in glutamatergic cerebellar granule cells, cerebellar astrocytes, and corresponding co‐cultures. It was evident that lactate is primarily a neuronal substrate and that lactate produced glycolytically from glucose in astrocytes serves as a substrate in neurons. Alanine was highly enriched with 13C in the neurons, whereas this was not the case in the astrocytes. Moreover, the cellular content and the amount of alanine released into the medium were higher in neurons than astrocytes. On incubation of the different cell types in medium containing alanine (1 mM), the astrocytes exhibited the highest level of accumulation. Altogether, these results indicate a preferential synthesis and release of alanine in glutamatergic neurons and uptake in cerebellar astrocytes. A new functional role of alanine may be suggested as a carrier of nitrogen from glutamatergic neurons to astrocytes, a transport that may operate to provide ammonia for glutamine synthesis in astrocytes and dispose of ammonia generated by the glutaminase reaction in glutamatergic neurons. Hence, a model of a glutamate‐glutamine/lactate‐alanine shuttle is presented. To elucidate if this hypothesis is compatible with the pattern of alanine metabolism observed in the astrocytes and neurons from cerebellum, the cells were incubated in a medium containing [15N]alanine (1 mM) and [5‐15N]glutamine (0.5 mM), respectively. Additionally, neurons were incubated with [U‐13C]glutamine to estimate the magnitude of glutamine conversion to glutamate. Alanine was labeled from [5‐15N]glutamine to 3.3% and [U‐13C]glutamate generated from [U‐13C]glutamine was labeled to 16%. In spite of the modest labeling in alanine, it is clear that nitrogen from ammonia is transferred to alanine via transamination with glutamate formed by reductive amination of α‐ketoglutarate. With regard to the astrocytic part of the shuttle, glutamine was labeled to 22% in one nitrogen atom whereas 3.2% was labeled in two when astrocytes were incubated in [15N]alanine. Moreover, in co‐cultures, [U‐13C]alanine labeled glutamate and glutamine equally, whereas [U‐13C]lactate preferentially labeled glutamate. Altogether, these results support the role proposed above of alanine as a possible ammonia nitrogen carrier between glutamatergic neurons and surrounding astrocytes and they show that lactate is preferentially metabolized in neurons and alanine in astrocytes.
Adenosine A1 Receptor in Cultured Neurons from Rat Cerebral Cortex
Authors: Ruiz María Angeles , Escriche Marisol , Lluis Carmen , Franco Rafael , Martín Mairena , Andrés Antonio , Ros Manuel
Published: March 2003

Adenosine A1 receptors (A1Rs) have been characterized

in primary cultures of neurons from cerebral cortex. The specific adenosine

A1 antagonist 8‐cyclopentyl‐1,3‐[3H]dipropylxanthine

bound to both membranes and intact cells. When saturation experiments were

performed in membranes, a KD value of 0.76 nM and

a Bmax of 57 fmol/mg of protein were obtained. Competition

assays revealed a pharmacological profile characteristic of A1Rs.

The presence of this receptor was further confirmed by RT‐PCR analysis. The

expression of the receptor showed no significant changes during the period of

culture studied, up to 12 days in vitro. A1R agonist inhibited

forskolin‐stimulated adenylyl cyclase, showing the functional coupling of

these receptors with the effector. αGi1,2 protein level,

detected by immunoblot, presented an increase during the period of culture.

This increase correlated with an increase in the mRNA level of

αGi1 but not αGi2. By immunochemical assays,

it is shown that these receptors are expressed in both the neuronal cell body

and the proximal dendrites. Colocalization of A1Rs with

microtubule‐associated protein 2 and cell surface adenosine deaminase was

shown by confocal microscopy. The high degree of colocalization observed

between A1Rs and ectoadenosine deaminase in neurons could suggest an important role of the enzyme in adenosine‐mediated neuromodulation.
Social Isolation‐Induced Decreases in Both the Abundance of Neuroactive Steroids and GABAA Receptor Function in Rat Brain
Authors: Serra Mariangela , Pisu Maria Giuseppina , Littera Martino , Papi Giacomo , Sanna Enrico , Tuveri Francesca , Usala Luca , Purdy Robert H , Biggio Giovanni
Published: March 2003

The effects of social isolation on behavior, neuroactive steroid concentrations, and GABAA receptor function were investigated in

rats. Animals isolated for 30 days immediately after weaning exhibited an

anxiety‐like behavioral profile in the elevated plus‐maze and Vogel conflict

tests. This behavior was associated with marked decreases in the

cerebrocortical, hippocampal, and plasma concentrations of pregnenolone,

progesterone, allopregnanolone, and allotetrahydrodeoxycorticosterone compared

with those apparent for group‐housed rats ; in contrast, the plasma

concentration of corticosterone was increased in the isolated animals. Acute

footshock stress induced greater percentage increases in the cortical

concentrations of neuroactive steroids in isolated rats than in group‐housed

rats. Social isolation also reduced brain GABAA receptor function,

as evaluated by measuring both GABA‐evoked Cl‐ currents in

Xenopus oocytes expressing the rat receptors and

tert‐[35S]butylbicyclophosphorothionate

([35S]TBPS) binding to rat brain membranes. Whereas the amplitude

of GABA‐induced Cl‐ currents did not differ significantly between

group‐housed and isolated animals, the potentiation of these currents by

diazepam was reduced at cortical or hippocampal GABAA receptors

from isolated rats compared with that apparent at receptors from group‐housed

animals. Moreover, the inhibitory effect of

ethyl‐β‐carboline‐3‐carboxylate, a negative allosteric modulator of

GABAA receptors, on these currents was greater at cortical

GABAA receptors from socially isolated animals than at those from

group‐housed rats. Finally, social isolation increased the extent of

[35S]TBPS binding to both cortical and hippocampal membranes. The

results further suggest a psychological role for neurosteroids and

GABAA receptors in the modulation of emotional behavior and mood.
Tissue Distribution, Autoradiography, and Metabolism of 4‐(2′‐Methoxyphenyl)‐1‐[2′ ‐[N‐2″‐Pyridinyl)‐p‐[18F]Fluorobenzamido]ethyl]piperazine (p‐[18F]MPPF), a New Serotonin 5‐HT1A Antagonist for Positron Emission Tomography
Authors: Plenevaux A , Weissmann D , Aerts J , Lemaire C , Brihaye C , Degueldre C , Le Bars D , Comar D , Pujol J ‐F , Luxen A
Published: March 2003

The in vivo behavior of 4‐(2′‐methoxyphenyl)‐1‐[2′‐[N‐(2″‐pyridinyl)‐p‐[18F]fluorobenzamido]ethyl]‐piperazine

(p‐[18F]MPPF), a new serotonin 5‐HT1A

antagonist, was studied in awake, freely moving rats. Biodistribution studies

showed that the carbon‐fluorine bond was stable in vivo, that this compound

was able to cross the blood‐brain barrier, and that a general diffusion

equilibrium could account for the availability of the tracer. The great

quantity of highly polar metabolites found in plasma did not contribute to the

small amounts of metabolites found in hippocampus, frontal cortex, and

cerebellum. Exvivo p‐[18F]MPPF and in vitro

8‐hydroxy‐2‐(di‐n‐[3H]propylamino)tetralin autoradiography

were compared both qualitatively and quantitatively. Qualitative evaluation

proved that the same brain regions were labeled and that the

p‐[18F]MPPF labeling is (a) in total agreement with the

known distribution of 5‐HT1A receptors in rats and (b)

characterized by very low nonspecific binding. Quantitative comparison

demonstrated that the in vivo labeling pattern obtained with

p‐[18F]MPPF cannot be explained by differences in regional

blood flow, capillary density, or permeability. The 5‐HT1A

specificity of p‐[18F]MPPF and binding reversibility were

confirmed in vivo with displacement experiments. Thus, this compound can be

used to evaluate parameters characterizing 5‐HT1A binding sites in the brain.
Early Alterations in Gene Expression and Cell Morphology in a Mouse Model of Huntington's Disease
Authors: Iannicola C , Moreno S , Oliverio S , Nardacci R , Ciofi‐Luzzatto A , Piacentini M
Published: March 2003

Several mouse models for Huntington's disease (HD) have been produced to date. Based on differences in strain, promoter, construct, and number of glutamines, these models have provided a broad spectrum of neurological symptoms, ranging from simple increases in aggressiveness with no signs of neuropathology, to tremors and seizures in absence of degeneration, to neurological symptoms in the presence of gliosis and TUNEL (terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling) positivity, and finally to selective striatal damage associated with electrophysiological and behavioral abnormalities. We decided to analyze the morphology of striatum and hippocampus from a mouse transgenic line obtained by microinjection of exon 1 from the HD gene after introduction of a very high number of CAG repeat units. We found a massive darkening and compacting of striatal and hippocampal neurons in affected mice, associated with a lower degree of more classical apoptotic cell condensation. We then explored whether this morphology could be explained with alterations in gene expression by hybridizing normal and affected total brain RNA to a panel of 588 known mouse cDNAs. We show that some genes are significantly and consistently up‐regulated and that others are down‐regulated in the affected brains. Here we discuss the possible significance of these alterations in neuronal morphology and gene expression.
Phospholipase D2 Activity Suppresses Hydrogen Peroxide‐Induced Apoptosis in PC12 Cells
Authors: Lee Sang Do , Lee Byoung Dae , Han Jung Min , Kim Jae Ho , Kim Yong , Suh Pann‐Ghill , Ryu Sung Ho
Published: March 2003

Abstract: Phospholipase D (PLD) plays an important role as an

effector in the membrane lipid‐mediated signal transduction. However, the

precise physiological functions of PLD are not yet well understood. In this

study, we examined the role of PLD activity in hydrogen peroxide

(H2O2)‐induced apoptosis in rat pheochromocytoma (PC12)

cells. Treatment of PC12 cells with H2O2 resulted in

induction of apoptosis in these cells, which is accompanied by the activation

of PLD. This H2O2‐induced apoptosis was enhanced

remarkably when phosphatidic acid production by PLD was selectively inhibited

by pretreating the PC12 cells with 1‐butanol. Expression of PLD2, but not of

PLD1, correlated with increased H2O2‐induced PLD

activity in a concentration‐ and time‐dependent manner. Concomitant with PLD

activation, the PLD2 activity suppressed H2O2‐induced

apoptosis in PC12 cells. Expression of PLD2 lipase‐inactive mutant (K758R) had

no effect on either PLD activity or apoptosis. PLD2 activity also suppressed

H2O2‐induced cleavage and activation of caspase‐3. Taken

together, the results suggest that PLD2 activity is specifically up‐regulated

by H2O2 in PC12 cells and that it plays a suppressive

role in H2O2‐induced apoptosis.
Characterization of Copper Interactions with Alzheimer Amyloid β Peptides
Authors: Atwood Craig S. , Scarpa Richard C. , Huang Xudong , Moir Robert D. , Jones Walton D. , Fairlie David P. , Tanzi Rudolph E. , Bush Ashley I.
Published: March 2003

Abstract: Cu and Zn have been shown to accumulate in the brains of

Alzheimer's disease patients. We have previously reported that Cu2+

and Zn2+ bind amyloid β (Aβ), explaining their enrichment

in plaque pathology. Here we detail the stoichiometries and binding affinities

of multiple cooperative Cu2+‐binding sites on synthetic Aβ1‐40

and Aβ1‐42. We have developed a ligand displacement technique

(competitive metal capture analysis) that uses metal‐chelator complexes to

evaluate metal ion binding to Aβ, a notoriously self‐aggregating peptide.

This analysis indicated that there is a very‐high‐affinity

Cu2+‐binding site on Aβ1‐42 (log Kapp =

17.2) that mediates peptide precipitation and that the tendency of this

peptide to self‐aggregate in aqueous solutions is due to the presence of trace

Cu2+ contamination (customarily ∼0.1 μM). In

contrast, Aβ1‐40 has much lower affinity for Cu2+ at this site

(estimated log Kapp = 10.3), explaining why this peptide

is less self‐aggregating. The greater Cu2+‐binding affinity of

Aβ1‐42 compared with Aβ1‐40 is associated with significantly

diminished negative cooperativity. The role of trace metal contamination in

inducing Aβ precipitation was confirmed by the demonstration that Aβ

peptide (10 μM) remained soluble for 5 days only in the presence

of high‐affinity Cu2+‐selective chelators.
Structure of the Mouse Glutamate Decarboxylase 65 Gene and Its Promoter
Authors: Makinae Kimitoshi , Kobayashi Takashi , Kobayashi Takayasu , Shinkawa Hideichi , Sakagami Hiroyuki , Kondo Hisatake , Tashiro Fumi , Miyazaki Jun‐ichi , Obata Kunihiko , Tamura Shinri , Yanagawa Yuchio
Published: March 2003

Abstract: GABA is synthesized by glutamate decarboxylase (GAD),
which has two forms, GAD65 and GAD67. To elucidate the molecular mechanisms of
mouse GAD65 (mGAD65) gene expression, we isolated and characterized the mGAD65
gene. The mGAD65 gene was found to be divided into 16 exons and spread over 75
kb. The sequence of the first exon and the 5′‐flanking region indicated
the presence of potential neuron‐specific cis‐regulatory elements. We
used transgenic mice to examine the expression pattern conferred by a 9.2‐kb
promoter‐proximal DNA fragment of the mGAD65 gene fused to the bacterial
lacZ reporter gene. Transgenic mice showed high β‐galactosidase activity specifically in brain and testis. They also showed characteristic patterns of transgene expression in olfactory bulb, cerebellar cortex, and spinal cord, a similar expression pattern to that of endogenous mGAD65. However, no transgene expression was observed in the ventral thalamus or hypothalamus, in which high mGAD65 gene expression levels have been observed. These results suggest that the 9.2‐kb DNA fragment of the mGAD65 gene is associated with its tissue‐specific expression and its targeted expression in GABAergic neurons of specific brain regions but that additional regulatory elements are necessary to obtain fully correct expression.
Nerve Tissue‐Specific (GLUD2) and Housekeeping (GLUD1) Human Glutamate Dehydrogenases Are Regulated by Distinct Allosteric Mechanisms
Authors: Plaitakis Andreas , Metaxari Maria , Shashidharan P.
Published: March 2003

Abstract: Human glutamate dehydrogenase (GDH), an enzyme central to
the metabolism of glutamate, is known to exist in housekeeping and nerve
tissue‐specific isoforms encoded by the GLUD1 and GLUD2
genes, respectively. As there is evidence that GDH function in vivo is
regulated, and that regulatory mutations of human GDH are associated with
metabolic abnormalities, we sought here to characterize further the functional
properties of the two human isoenzymes. Each was obtained in recombinant form
by expressing the corresponding cDNAs in Sf9 cells and studied with respect to
its regulation by endogenous allosteric effectors, such as purine nucleotides
and branched chain amino acids. Results showed that L‐leucine, at 1.0
mM, enhanced the activity of the nerve tissue‐specific
(GLUD2‐derived) enzyme by ∼1,600% and that of the
GLUD1‐derived GDH by ∼75%. Concentrations of L‐leucine similar to
those present in human tissues (∼0.1 mM) had little effect on
either isoenzyme. However, the presence of ADP (10‐50 μM)
sensitized the two isoenzymes to L‐leucine, permitting substantial enzyme
activation at physiologically relevant concentrations of this amino acid.
Nonactivated GLUD1 GDH was markedly inhibited by GTP (IC50
= 0.20 μM), whereas nonactivated GLUD2 GDH was totally
insensitive to this compound (IC50 > 5,000 μM). In
contrast, GLUD2 GDH activated by ADP and/or L‐leucine was amenable to
this inhibition, although at substantially higher GTP concentrations than the
GLUD1 enzyme. ADP and L‐leucine, acting synergistically, modified the
cooperativity curves of the two isoenzymes. Kinetic studies revealed
significant differences in the Km values obtained for
α‐ketoglutarate and glutamate for the GLUD1‐ and the
GLUD2‐derived GDH, with the allosteric activators differentially altering these values. Hence, the activity of the two human GDH is regulated by distinct allosteric mechanisms, and these findings may have implications for the biologic functions of these isoenzymes.
Polyamine Uptake in Cultured Astrocytes
Authors: Dot Josep , Lluch Mónica , Blanco Isaac , Rodríguez‐Alvarez José
Published: March 2003

Abstract: The properties and regulation of the polyamine transport
system in brain are still poorly understood. The present study shows, for the
first time, the existence of a polyamine transport system in cerebellar
astrocytes and suggests that polyamine uptake is mediated by a single and
saturable high‐affinity transport system for putrescine, spermine, and
spermidine (Km = 3.2, 1.2, and 1.8 μM,
respectively). Although substitution of NaCl by choline chloride produced a
decrease in the putrescine, spermine, and spermidine uptake, it seems that
polyamine transport in cerebellar astrocytes is not mediated by an
Na+ cotransport as in the presence of Na+ and cholinium,
polyamine uptake was much lower than when measured in a sucrose‐based medium.
On the other hand, ouabain, gramicidin (a Na+ ionophore), and
ionomycin (a Ca2+ ionophore) produced a strong inhibition of
polyamine uptake, suggesting that membrane potential could have an important
role in the functioning of the astroglial polyamine uptake system. Moreover,
protein kinase C inhibition produced an enhancement of polyamine uptake,
whereas stimulation of protein kinase C with phorbol esters inhibited
polyamine uptake. Alternatively, the tyrosine kinase inhibitor genistein
caused a marked reduction in the uptake. No effects on polyamine uptake were
observed with inhibitors and activators of cyclic AMP‐dependent protein kinase
or when Ca2+/calmodulin‐dependent protein kinase II was inhibited with KN‐62. These results suggest that the polyamine uptake system in cerebellar astrocytes could be modulated by protein kinase C and tyrosine kinase activities.
Transcriptional Regulation of 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase Gene Expression by Cyclic AMP in C6 Cells
Authors: Gravel M. , Gao E. , Hervouet‐Zeiber C. , Parsons V. , Braun P. E.
Published: March 2003

Abstract: It was recently shown that the two transcripts encoding the isoforms of 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP1 and CNP2) are differentially regulated during the process of oligodendrocyte maturation. In oligodendrocyte precursors, only CNP2 mRNA is present, whereas in differentiating oligodendrocytes, both CNP1 and CNP2 mRNAs are expressed. This pattern of CNP expression is likely due to stage‐specific transcriptional regulation of the two CNP promoters during the process of oligodendrocyte differentiation. Here, we report the influence of increased intracellular cyclic AMP (cAMP) levels on the transcription of both CNP1 and CNP2 mRNAs in rat C6 glioma cells. We found that the transcription of CNP1 mRNA was significantly increased in comparison with that of CNP2 mRNA in cells treated with cAMP analogues to elevate intracellular cAMP levels. This up‐regulation of CNP1 expression (a) is due to an increase of transcription, (b) requires de novo protein synthesis, and (c) requires the activity of protein kinase A. These results are physiologically significant and support the idea that a cAMP‐mediated pathway is part of the molecular mechanisms regulating the expression of CNP1 in oligodendrocytes. The regulation of CNP1 promoter activity by cAMP was then investigated in stably transfected C6 cell lines containing various deletions of the CNP promoter directing the bacterial chloramphenicol acetyltransferase gene. We showed that the sequence between nucleotides ‐126 and ‐102 was essential for the cAMP‐dependent induction of CNP1 expression. Gel retardation analysis showed that two protein‐DNA complexes are formed between this sequence and nuclear factors from C6 cells treated or not treated with cAMP. This suggests that the induction of CNP1 mRNA transcription is not mediated by changes in binding of nuclear factors that interact directly with the ‐126/‐102 sequence. Sequence analysis of this region revealed the presence of a putative activator protein‐2 (AP‐2) binding site. It is interesting that mutagenesis of this region resulted in a significant reduction in transcriptional responses to cAMP, implying a possible role for the AP‐2 factor in the expression of CNP1. In addition, we have shown that putative binding sites for activator protein‐4 and nuclear factor‐1 adjacent to the AP‐2 site are required for efficient induction of CNP1 expression by cAMP. Taken together, our results show that the cAMP‐dependent accumulation of CNP1 mRNA appears to depend on the synergistic interaction of several regulatory elements.
RGS7 Is Palmitoylated and Exists as Biochemically Distinct Forms
Authors: Rose Jeremy J. , Taylor Jackie B. , Shi Jing , Cockett Mark I. , Jones Philip G. , Hepler John R.
Published: March 2003

Abstract:
Regulator of G protein
signaling (RGS) proteins are GTPase‐activating proteins that
modulate neurotransmitter and G protein signaling. RGS7 and its binding
partners Gα and Gβ5 are enriched in brain, but biochemical
mechanisms governing RGS7/Gα/Gβ5 interactions and membrane
association are poorly defined. We report that RGS7 exists as one cytosolic
and three biochemically distinct membrane‐bound fractions (salt‐extractable,
detergent‐extractable, and detergent‐insensitive) in brain. To define factors
that determine RGS7 membrane attachment, we examined the biochemical
properties of recombinant RGS7 and Gβ5 synthesized in Spodoptera
frugiperda insect cells. We have found that membrane‐bound but not
cytosolic RGS7 is covalently modified by the fatty acid palmitate. Gβ5 is
not palmitoylated. Both unmodified (cytosolic) and palmitoylated
(membrane‐derived) forms of RGS7, when complexed with Gβ5, are equally
effective stimulators of Gαo GTPase activity, suggesting that
palmitoylation does not prevent RGS7/Gαo interactions. The
isolated core RGS domain of RGS7 selectively binds activated
Gαi/o in brain extracts and is an effective stimulator of
both Gαo and Gαi1 GTPase activities in
vitro. In contrast, the RGS7/Gβ5 complex selectively interacts with
Gαo only, suggesting that features outside the RGS domain and/or Gβ5 association dictate RGS7‐Gα interactions. These findings define previously unrecognized biochemical properties of RGS7, including the first demonstration that RGS7 is palmitoylated.
Altered Serotonin and Dopamine Metabolism in the CNS of Serotonin 5‐HT1A or 5‐HT1B Receptor Knockout Mice
Authors: Ase Ariel R. , Reader Tomás A. , Hen René , Riad Mustapha , Descarries Laurent
Published: March 2003

Abstract: Measurements of serotonin (5‐HT), dopamine (DA), andnoradrenaline, and of 5‐HT and DA metabolites, were obtained by HPLC from 16brain regions and the spinal cord of 5‐HT1A or 5‐HT1Bknockout and wild‐type mice of the 129/Sv strain. In 5‐HT1Aknockouts, 5‐HT concentrations were unchanged throughout, but levels of 5‐HTmetabolites were higher than those of the wild type in dorsal/medial raphenuclei, olfactory bulb, substantia nigra, and locus coeruleus. This was takenas an indication of increased 5‐HT turnover, reflecting an augmented basalactivity of midbrain raphe neurons and consequent increase in theirsomatodendritic and axon terminal release of 5‐HT. It provided a likelyexplanation for the increased anxious‐like behavior observed in5‐HT1A knockout mice. Concomitant increases in DA content and/or DAturnover were interpreted as the result of a disinhibition of DA, whereasincreases in noradrenaline concentration in some territories of projection ofthe locus coeruleus could reflect a diminished activity of its neurons. In5‐HT1B knockouts, 5‐HT concentrations were lower than those of thewild type in nucleus accumbens, locus coeruleus, spinal cord, and probablyalso several other territories of 5‐HT innervation. A decrease in DA,associated with increased DA turnover, was measured in nucleus accumbens.These changes in 5‐HT and DA metabolism were consistent with the increasedaggressiveness and the supersensitivity to cocaine reported in5‐HT1B knockout mice. Thus, markedly different alterations in CNSmonoamine metabolism may contribute to the opposite behavioral phenotypes ofthese two knockouts.
Levels, Metabolism, and Pharmacological Activity of Anandamide in CB1 Cannabinoid Receptor Knockout Mice
Authors: Di Marzo Vincenzo , Breivogel Chris S. , Tao Qing , Bridgen David T. , Razdan Raj K. , Zimmer Anne M. , Zimmer Andreas , Martin Billy R.
Published: March 2003

Abstract: Anandamide [arachidonylethanolamide (AEA)] appears to be an endogenous agonist of brain cannabinoid receptors (CB1), yet some of the neurobehavioral effects of this compound in mice are unaffected by a selective CB1 antagonist. We studied the levels, pharmacological actions, and degradation of AEA in transgenic mice lacking the CB1 gene. We quantified AEA and the other endocannabinoid, 2‐arachidonoyl glycerol, in six brain regions and the spinal cord by isotope‐dilution liquid chromatography‐mass spectrometry. The distribution of endocannabinoids and their inactivating enzyme, fatty acid amide hydrolase, were found to overlap with CB1 distribution only in part. In CB1 knockout homozygotes (CB1‐/‐), the hippocampus and, to a lesser extent, the striatum exhibited lower AEA levels as compared with wild‐type (CB1+/+) controls. These data suggest a ligand/receptor relationship between AEA and CB1 in these two brain regions, where tonic activation of the receptor may tightly regulate the biosynthesis of its endogenous ligand. 2‐Arachidonoyl glycerol levels and fatty acid amide hydrolase activity were unchanged in CB1‐/‐ with respect to CB1+/+ mice in all regions. AEA and Δ9‐tetrahydrocannabinol (THC) were tested in CB1‐/‐ mice for their capability of inducing analgesia and catalepsy and decreasing spontaneous activity. The effects of AEA, unlike THC, were not decreased in CB1‐/‐ mice. AEA, but not THC, stimulated GTPγS binding in brain membranes from CB1‐/‐ mice, and this stimulation was insensitive to CB1 and CB2 antagonists. We suggest that non‐CB1, non‐CB2 G protein‐coupled receptors might mediate in mice some of the neuro‐behavioral actions of AEA.
Glutamine Synthetase and Glutamine Synthetase‐Like Protein from Human Brain
Authors: Boksha Irina S. , Tereshkina Elena B. , Burbaeva Gulnur Sh.
Published: March 2003

Abstract: Glutamine synthetase (GS; EC 6.3.1.2), a key enzyme ofglutamate metabolism, and another enzyme possessing highhydroxylamine‐L‐glutamine transferase activity comparable to that of GS andtermed GS‐like protein (GSLP) were purified from human brain concurrently. Intwo‐dimensional electrophoresis, GS subunits migrate to at least six differentpositions (44 ± 1 kDa, pl = 6.4‐6.7), whereas GSLP subunits migrate toat least four different positions (54 ± 1 kDa, pl = 5.9‐6.2).Dependences of enzymatic activity in the transferase reaction onconcentrations of Mn2+ and Mg2+ for GS and GSLP aredifferent. High immunological cross‐reactivity between GS and GSLP wasobserved in ELISA. Nevertheless, antisera were raised to GS and GSLP, and amethod was developed for the separate detection of GS and GSLP in brainextracts by enzyme‐chemiluminescent amplified (ECL) immunoblotting. Thedistribution of GS and GSLP immunoreactivities between soluble protein andcrude mitochondrial fractions indicates tighter association with theparticulate fraction for GSLP than for GS. The results from activitymeasurements suggest that the hydroxylamine‐L‐glutamine transferase activitymeasured routinely in protein extracts from brain is the sum of GS and GSLPactivities. Similarly, immunoreactivity evaluated by ELISA is a sum ofimmunoreactivities of GS and GSLP. The relative contributions of GS and GSLPto the total immunoreactivity can be evaluated by ECL‐immunoblotting.
Structure of the Human Serotonin 5‐HT4 Receptor Gene and Cloning of a Novel 5‐HT4 Splice Variant
Authors: Bender Eckhard , Pindon Armelle , Van Oers Irma , Zhang Yu‐Bin , Gommeren Walter , Verhasselt Peter , Jurzak Mirek , Leysen Josee , Luyten Walter
Published: March 2003

Abstract: Several variants of the serotonin 5‐HT4 receptor are known to be produced by alternative splicing. To survey the existence and usage of exons in humans, we cloned the human 5‐HT4 gene. Based on sequence analysis seven C‐terminal variants (a‐g) and one internal splice variant (h) were found. We concentrated in this study on the functional characterization of the novel splice variant h, which leads to the insertion of 14 amino acids into the second extracellular loop of the receptor. The h variant was cloned as a splice combination with the C‐terminal b variant; therefore, we call this receptor 5‐HT4(hb). This novel receptor variant was expressed transiently in COS‐7 cells, and its pharmacological profile was compared with those of the previously cloned 5‐HT4(a) and 5‐HT4(b) isoforms, with the latter being the primary reference for the h variant. In competition binding experiments using reference 5‐HT4 ligands, no significant differences were detected. However, the broadly used 5‐HT4 antagonist GR113808 discriminated functionally among the receptor variants investigated. As expected, it was an antagonist on the 5‐HT4(a) and 5‐HT4(b) variant but showed partial agonistic activity on the 5‐HT4(hb) variant. These data emphasize the importance of variations introduced by splicing for receptor pharmacology and may help in the understanding of conflicting results seen with 5‐HT4 ligands in different model systems.
Mitogenic Signaling via Endogenous κ‐Opioid Receptors in C6 Glioma Cells
Authors: Bohn Laura M. , Belcheva Mariana M. , Coscia Carmine J.
Published: March 2003

Abstract: As reports on G protein‐coupled receptor signal transduction mechanisms continue to emphasize potential differences in signaling due to relative receptor levels and cell type specificities, the need to study endogenously expressed receptors in appropriate model systems becomes increasingly important. Here we examine signal transduction mechanisms mediated by endogenous κ‐opioid receptors in C6 glioma cells, an astrocytic model system. We find that the κ‐opioid receptor‐selective agonist U69,593 stimulates phospholipase C activity, extracellular signal‐regulated kinase 1/2 phosphorylation, PYK2 phosphorylation, and DNA synthesis. U69,593‐stimulated extracellular signal‐regulated kinase 1/2 phosphorylation is shown to be upstream of DNA synthesis as inhibition of signaling components such as pertussis toxin‐sensitive G proteins, L‐type Ca2+ channels, phospholipase C, intracellular Ca2+ release, protein kinase C, and mitogen‐activated protein or extracellular signal‐regulated kinase kinase blocks both of these downstream events. In addition, by overexpressing dominant‐negative or sequestering mutants, we provide evidence that extracellular signal‐regulated kinase 1/2 phosphorylation is Ras‐dependent and transduced by Gβγ subunits. In summary, we have delineated major features of the mechanism of the mitogenic action of an agonist of the endogenous κ‐opioid receptor in C6 glioma cells.
Role of Striatal Serotonin2A and Serotonin2C Receptor Subtypes in the Control of In Vivo Dopamine Outflow in the Rat Striatum
Authors: Lucas Guillaume , Spampinato Umberto
Published: March 2003

Abstract: This study investigated, using in vivo microdialysis in the striatum of freely moving rats, the role of striatal serotonin2A (5‐HT2A) and 5‐HT2C receptor subtypes in the modulation of dopamine (DA) and 3, 4‐dihydroxyphenylacetic acid (DOPAC) outflow, both in basal conditions and under activation induced by subcutaneous administration of 0.01 mg/kg haloperidol. The different 5‐HT2 agents used were applied intrastriatally at a 1 μM concentration through the microdialysis probe. Basal DA efflux was enhanced (27%) by the 5‐HT2A/2B/2C agonist 1‐(4‐iodo‐2,5‐dimethoxyphenyl)‐2‐aminopropane (DOI) and reduced (‐30%) by the 5‐HT2B/2C antagonist SB 206553. It was unaffected by infusion of the 5‐HT2A antagonist SR 46349B. The effect of DOI was abolished by SB 206553 but not modified by SR 46349B. Haloperidol‐stimulated DA efflux (65‐70%) was reduced by both SR 46349B (‐32%) and the 5‐HT2A/2B/2C antagonist ritanserin (‐30%) but not affected by SB 206553. Conversely, the effect of haloperidol was potentiated (22%) when DOI was coperfused with SB 206553. Also, haloperidol‐stimulated DOPAC outflow (40‐45%) was reduced (‐20%) by SR 46349B and potentiated (25%) by the combination of SB 206553 with DOI. These results indicate that striatal 5‐HT2A receptors, probably through activation of DA synthesis, positively modulate DA outflow only under activated conditions. In contrast, striatal 5‐HT2C receptors exert a facilitatory control on basal DA efflux, which appears to be both tonic and phasic.
Visualization of the Vesicular Acetylcholine Transporter in Living Cholinergic Cells
Authors: Santos M. S. , Prado V. F. , Barbosa J. , Kushmerick C. , Gomez M. V. , Prado M. A. M.
Published: March 2003

We regret that we must retract the article Visualization of the Vesicular Acetylcholine Transporter in Living Cholinergic Cells by M. S. Santos, J. Barbosa Jr., C. Kushmerick, M. V. Gomez, V. F. Prado, and M. A. M. Prado (J. Neurochem. 74, 2425‐2435, 2000). We have made an unintentional mistake in the construction of the enhanced green fluorescent protein (EGFP) vectors, and consequently the vesicular acetylcholine transporter (VAChT) and its truncated forms are incorrectly expressed. We have repeated the key experiments with proper constructs and have found that the expression pattern is clearly different from that reported in the article. The truncated form of VAChT, without the C‐terminal tail, presents a distinct pattern of expression when compared to VAChT, and we have found no evidence that the C‐terminal tail of VAChT is able to drive EGFP to varicosity sites. As a consequence of this problem, our earlier conclusions were incorrect. We apologize for this mistake and for any problems that we may have caused.