bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction Between the Serotoninergic and Dopaminergic Systems in d‐Fenfluramine‐Induced Activation of c‐fos and junB Genes in Rat Striatal Neurons
Authors: Gardier A. M. , Moratalla R. , Cuéllar B. , Sacerdote M. , Guibert B. , Lebrec H. , Graybiel A. M.
Published: March 2003

Abstract: To test for the relative contributions of the dopaminergic
and serotoninergic systems in the striatum to the effects of
d‐fenfluramine, an indirect serotonin receptor agonist, we assessed
the expression of Fos/Jun proteins induced by d‐fenfluramine given
alone or in the presence of dopaminergic or serotoninergic agents. To
determine the neuronal targets of d‐fenfluramine in the striatum, we
identified the phenotypes of striatal neurons in which d‐fenfluramine
induced Fos expression. Our results demonstrated that d‐fenfluramine
evokes nuclear expression of Fos/Jun B proteins in the striatum, and that the
Fos expression was dose‐dependent and accompanied by transient induction of
c‐fos mRNA. Fos expression was blocked by
p‐chloroamphetamine, a serotoninergic neurotoxin. Pretreatment with
SCH 23390, a D1‐dopamine receptor antagonist, led to a marked
decrease in Fos/Jun B expression in the caudoputamen, but not in the cortex,
whereas pretreatment with methiothepin, a nonselective serotonin
5‐HT1 receptor antagonist, blocked Fos expression completely in the
cortex and only partially in the caudoputamen. The expression of Fos/Jun B in
the striatum occurred mainly in dynorphin‐containing neurons and in a
subpopulation of striatal interneurons that exhibited NADPH‐diaphorase
activity. Most of the enkephalin‐containing neurons of the striatum did not
show Fos/Jun B staining. These results suggest that the mechanism by which
d‐fenfluramine induces c‐fos and jun B expression in the rat caudoputamen depends at least in part on activation of the dopaminergic system by serotonin.
Possible Role of CREB in the Stimulation of Oligodendrocyte Precursor Cell Proliferation by Neurotrophin‐3
Authors: Johnson Jamie R. , Chu Annie K. , Sato‐Bigbee Carmen
Published: March 2003

Abstract:We have previously shown that the transcription factor CREB (cyclic AMP‐response element binding protein) could be a mediator of neuronal signals that, coupled to different signal transduction pathways, may play different regulatory roles at specific stages of oligodendrocyte (OLG) development. We have found before that in committed OLGs, CREB activation by phosphorylation can be triggered by β‐adrenergic stimulation and appears to play a role in the induction of OLG differentiation by cyclic AMP. In contrast, in OLG precursor cells, CREB phosphorylation is stimulated by neuroligands that increase calcium levels by a process that involves a mitogen‐activated protein kinase (MAPK)/protein kinase C (PKC) pathway. This observation suggested that at this early developmental stage, CREB could play a role in regulating cell proliferation. In support of this hypothesis, we have now found that a rapid and dramatic stimulation of CREB phosphorylation is one of the earliest events that precedes the increase in cell proliferation that is observed when OLG precursors are treated with neurotrophin‐3 (NT‐3). Experiments in which CREB phosphorylation was investigated in the presence of different kinase inhibitors indicated that the activation of this transcription factor in the presence of NT‐3 is mediated by the concerted action of MAPK‐ and PKC‐dependent signal transduction pathways. Moreover, our present results also showed that down‐regulation of CREB expression in the OLG precursors abolished the increase in DNA synthesis that is observed when the cultures are treated with NT‐3. Thus, these results support the idea that in immature OLG precursors, CREB plays an important role in transducing signals which, like NT‐3, may regulate cell proliferation.
Interaction of Human Substantia Nigra Neuromelanin with Lipids and Peptides
Authors: Zecca Luigi , Costi Patrizia , Mecacci Claudio , Ito Shosuke , Terreni Mariarosa , Sonnino Sandro
Published: March 2003

Abstract: Neuromelanin was isolated from human substantia nigra using different procedures. In the pigment isolated by any of these procedures a peptide component covalently bound to the melanic structure was found, as shown by treatment with reagents known to eliminate noncovalently bound proteins. The amino acid content of such a peptide component was reproducible and corresponded to ∼15% of the neuromelanin weight. Neuromelanin also showed the ability to absorb specifically lipid molecules, ∼20% of its weight, and among these lipids cholesterol was identified, constituting ∼5% of the total lipid mixture. A synthetic melanin, incubated with putamen homogenate, bound tissue peptides with an amino acid content quite close to that of neuromelanin. The same synthetic melanin adsorbed a lower amount of lipids from the putamen homogenate compared with neuromelanin. The sulfur content of neuromelanin was also reproducible even using different isolation procedures. A nonpigmented tissue like corpus callosum was used as a control and extracted by the method used for neuromelanin isolation; a total elimination of tissue components was found, thus demonstrating the capability of the reported procedures to isolate neuromelanin alone. The presence of a peptide component in the neuromelanin structure and the selective affinity for lipid molecules suggest new aspects of the functional role and metabolic pathway of neuromelanin.
Cloning of Rat Parkin cDNA and Distribution of Parkin in Rat Brain
Authors: Gu Wen‐Jie , Abbas Nacer , Lagunes Martin Zarate , Parent André , Pradier Laurent , Bohme Georg A. , Agid Yves , Hirsch Etienne C. , Raisman‐Vozari Rita , Brice Alexis
Published: March 2003

Abstract: The rat parkin cDNA sequence was characterized after
screening a rat hypothalamus cDNA library with a 32P‐labeled probe containing the entire open reading frame of the human parkin cDNA. This sequence encompasses 1,576 bp and contains a single open reading frame that encodes a 465‐amino acid protein. The rat parkin amino acid sequence exhibits a very striking homology to the human and mouse parkin, with 85 and 95% identity, respectively. Both the N‐terminal ubiquitin and the ring‐IBR (in between ring)‐ring finger domains appear to be highly conserved among rat, human, and mouse parkin. An affinity‐purified polyclonal antibody (ASP5p) was generated with a synthetic peptide corresponding to amino acids 295‐311 of the parkin sequence, which is identical in the three species. Western blotting revealed that ASP5p recognizes a single 52‐kDa band, which corresponds to the molecular mass of the parkin protein. Immunostaining with ASP5p showed that parkin is principally located in the cytoplasm of neurons that are widely distributed in the rat brain. Parkin‐immunoreactive neurons abound in structures that are specifically targeted in Parkinson's disease, e.g., subtantia nigra, but are also present in unaffected structures, e.g., cerebellum. Furthermore, parkin‐enriched glial cells can be detected in various nuclei of the rat brain. Thus, the role of parkin may be much more global than previously thought on the basis of genetic findings gathered in cases of early‐onset parkinsonism.
L‐Arginine Uptake and Release by Cultured Avian Retinal Cells
Authors: Cossenza Marcelo , De Carvalho Roberto Paes
Published: March 2003

Abstract: The availability of L‐arginine is of pivotal importance for the synthesis of nitric oxide, a signaling molecule in the CNS. Here we show the presence of a high‐affinity L‐arginine uptake system (Km of 4.4 ± 0.5 μM and a Vmax of 26.0 ± 0.9 fmol/well/min) in cultured chick retinal cells. Different compounds, such as NG‐mono‐methyl‐L‐arginine and L‐lysine, were able to inhibit the uptake that was also inhibited 60‐70% in the absence of sodium and/or calcium ions. No trans stimulation was observed when cells were preloaded with L‐lysine. The data indicate that the L‐arginine uptake in cultured retinal cells is partially mediated by the y+ system, but has a great contribution of the B0,+ system. Autoradiographic studies revealed that the uptake is predominant in glial cells and can also be detected in neurons, whereas immunocytochemistry of nitric oxide synthase and L‐citrulline showed that the enzyme is present in neurons and photoreceptors, but not in glial cells. L‐[3H]Arginine is released from purified glial cultures incubated with high concentrations of potassium in the extracellular medium. Moreover, the amino acid released from preloaded glial cells was taken up by purified neuronal cultures. These results indicate that L‐arginine released from glial cells is taken up by neurons and used as substrate for the synthesis of nitric oxide.
High Extracellular Potassium Modulates Nitric Oxide Synthase Expression in Human Astrocytes
Authors: Morgan Anna C. A. , Chang Hee‐Yoon , Liu Judy S. H. , Hua Liwei L. , Lee Sunhee C.
Published: March 2003

Abstract: Inducible nitric oxide synthase (iNOS) is a molecule of great interest, given the numerous biological activities of nitric oxide and the documented expression of iNOS in several CNS pathologies. There also appears to be species‐dependent regulation of iNOS expression as well as CNS‐specific regulation. In this study, we have examined cultures of cytokine‐activated primary human astrocytes as a model system with which to study the mechanisms of iNOS regulation in human CNS. As one of the major functions of astrocytes is spatial buffering of K+ ion, we examined the effect of high extracellular KCl on astrocyte iNOS expression. The results demonstrate that KCl at 25‐75 mM potently inhibits astrocyte nitrite production stimulated by interleukin‐1 (IL‐1)/interferon‐γ (IFNγ). In addition, several potassium channel inhibitors such as CsCl, tetraethylammonium, and 4‐aminopyridine as well as nigericin inhibited astrocyte iNOS expression induced by IL‐1/IFNγ. These results demonstrate a novel role for astrocyte potassium channel activity in modulation of astrocyte function. They further suggest neural‐specific mechanisms for glial iNOS regulation.
Tetanus Toxin Fragment C Binds to a Protein Present in Neuronal Cell Lines and Motoneurons
Authors: Herreros Judit , Lalli Giovanna , Montecucco Cesare , Schiavo Giampietro
Published: March 2003

Abstract:Tetanus neurotoxin is one of the most powerful protein toxins known, acting in vivo at femtomolar doses. Two main factors determine its high potency: a protease activity restricted to a single intracellular substrate and its absolute neurospecificity. Whereas the enzymatic properties of tetanus toxin have been thoroughly defined, the nature of its neuronal receptor(s) and their involvement in the intracellular trafficking of tetanus toxin are poorly understood. Using binding and crosslinking experiments, we report here on the characterisation of anN‐glycosylated 15‐kDa interacting protein, which behaves as an integral membrane protein. This putative receptor specifically interacts with the binding domain (fragment C) of tetanus toxin and not with several related botulinum neurotoxins in spinal cord motoneurons and neuronal‐like cell lines. Sialic acid‐specific lectins antagonise the binding of tetanus toxin to the cell surface and to the 15‐kDa protein, supporting the central role of sialic acid residues in the recognition process. Altogether, these results indicate the existence of a neuronal protein receptor for tetanus toxin whose identification is likely to constitute a key step in the analysis of the molecular machinery involved in the toxin internalisation and retrograde transport.
Characteristics of the Calcium‐Triggered Mitochondrial Permeability Transition in Nonsynaptic Brain Mitochondria
Authors: Kristián Tibor , Gertsch Jeff , Bates Timothy E. , Siesjö Bo K.
Published: March 2003

Abstract: The objective of the present study was to assess the capacity of nonsynaptic brain mitochondria to accumulate Ca2+ when subjected to repeated Ca2+ loads, and to explore under what conditions a mitochondrial permeability transition (MPT) pore is assembled. The effects of cyclosporin A (CsA) on Ca2+ accumulation and MPT pore assembly were compared with those obtained with ubiquinone 0 (Ub0), a quinone that is a stronger MPT blocker than CsA, when tested on muscle and liver mitochondria. When suspended in a solution containing phosphate (2 mM) and Mg2+ (1 mM), but no ATP or ADP, the brain mitochondria had a limited capacity to accumulate Ca2+ (210 nmol/mg of mitochondrial protein). Furthermore, when repeated Ca2+ pulses (40 nmol/mg of protein each) saturated the uptake system, the mitochondria failed to release the Ca2+ accumulated. However, in each instance, the first Ca2+ pulse was accompanied by a moderate release of Ca2+, a release that was not observed during the subsequent pulses. The initial release was accompanied by a relatively marked depolarization, and by swelling, as assessed by light‐scattering measurements. However, as the swelling was <50% of that observed following addition of alamethicin, it is concluded that the first Ca2+ pulse gives rise to an MPT in a subfraction of the mitochondrial population. CsA, an avid blocker of the MPT pore, only marginally increased the Ca2+‐sequestrating capacity of the mitochondria. However, CsA eliminated the Ca2+ release accompanying the first Ca2+ pulse. The effects of CsA were shared by Ub0, but when the concentration of Ub0 exceeded 20 μM, it proved toxic. The results thus suggest that brain mitochondria are different from those derived from a variety of other sources. The major difference is that a fraction of the brain mitochondria, studied presently, depolarized and showed signs of an MPT. This fraction, but not the remaining ones, contributed to the chemically and electron microscopically verified mitochondrial swelling.
Region‐Specific Enhancement of Basal Extracellular and Cocaine‐Evoked Dopamine Levels Following Constitutive Deletion if tge Seritibub1B Receptor
Authors: Shippenberg T. S. , Hen R. , He M.
Published: March 2003

Abstract : The behavioral effects of cocaine are enhanced following

constitutive deletion of the serotonin1B receptor. The neural

substrates mediating the enhanced response to cocaine are unknown. The present

studies determined whether basal dopamine dynamics or cocaine‐evoked dopamine

levels are altered in projection areas of mesostriatal or mesoaccumbens

dopamine neurons following serotonin1B receptor deletion. Male

wild‐type and serotonin1B knockout mice were implanted with

microdialysis guide cannulas aimed at the dorsal striatum or nucleus

accumbens. The zero net flux method of quantitative microdialysis was used to

quantify basal extracellular dopamine concentrations (DAext) and

the extraction fraction of dopamine (Ed), which provides

an index of dopamine uptake. Conventional microdialysis techniques were used

to quantify cocaine (0, 5.0, and 20.0 mg/kg)‐evoked dopamine overflow. Basal

DAext and Ed did not differ in striatum of

wild‐type and knockout mice. Similarly, cocaine‐stimulated dopamine overflow

did not differ between genotype. The basal Ed did not

differ in the nucleus accumbens of wild‐type and knockout mice. However,

DAext was significantly elevated in the nucleus accumbens of

knockout mice. Cocaine‐evoked dopamine overflow (nM) was also

enhanced in the nucleus accumbens of knockout mice. However, the

cocaine‐induced increase in dopamine levels, relative to basal values, did not

differ between genotype. These data demonstrate that deletion of the

serotonin1B receptor is associated with increases in basal

DAext in the nucleus accumbens. This increase is not associated

with an alteration in Ed, suggesting increased basal

dopamine release in these animals. It is hypothesized that these alterations

in presynaptic neuronal activity are a compensatory response to constitutive

deletion of the serotonin1B receptor and may contribute to the enhanced behavioral effects of psychostimulants observed in knockout mice.
Calcineurin Activity Is Regulated Both by Redox Compounds and by Mutant Familial Amyotrophic Lateral Sclerosis‐Superoxide Dismutase
Authors: Ferri Alberto , Gabbianelli Roberta , Casciati Arianna , Paolucci Egle , Rotilio Giuseppe , Carrì Maria Teresa
Published: March 2003

Calcineurin (CN) is a protein phosphatase involved in a wide range of cellular responses to calcium‐mobilizing signals, and a role for this enzyme in neuropathology has been postulated. We have investigated the possibility that redox modulation of CN activity is relevant to neuropathological conditions where an imbalance in reactive oxygen species has been described. We have monitored CN activity in cultured human neuroblastoma SH‐SY5Y cells and obtained evidence that CN activity is promoted by treatment with ascorbate or dithiothreitol and impaired by oxidative stress. Evidence for the existence of a redox regulation of this enzyme has been also obtained by overexpression of wild‐type antioxidant Cu,Zn superoxide dismutase (SOD1) that promotes CN activity and protects it from oxidative inactivation. On the contrary, overexpression of mutant SOD1s associated with familial amyotrophic lateral sclerosis (FALS) impairs CN activity both in transfected human neuroblastoma cell lines and in the motor cortex of brain from FALS‐transgenic mice. These data suggest that CN might be a target in the pathogenesis of SOD1‐linked FALS.
Phosphorylation and Activation of Brain Aromatic l‐Amino Acid Decarboxylase by Cyclic AMP‐Dependent Protein Kinase
Authors: Duchemin Anne‐Marie , Berry Mark D , Neff Norton H , Hadjiconstantinou Maria
Published: March 2003

Aromatic l‐amino acid decarboxylase (AAAD), an enzyme required

for the synthesis of catecholamines, indoleamines, and trace amines, is

rapidly activated by cyclic AMP‐dependent pathways in striatum and midbrain in

vivo, suggesting enzyme phosphorylation. We now report that the catalytic

subunit of cyclic AMP‐dependent protein kinase (PKA) directly phosphorylated

AAAD immunoprecipitated from homogenates prepared from the mouse striatum and

midbrain in vitro. Under the same phosphorylation conditions, the catalytic

subunit of PKA also phosphorylated a recombinant AAAD protein expressed in

Escherichia coli transfected with an AAAD cDNA isolated from the bovine adrenal gland. The PKA‐induced AAAD phosphorylation of immunoprecipitates from striatum and midbrain was time and concentration dependent and blocked by a specific PKA peptide inhibitor. Incubation of the catalytic subunit of PKA with striatal homogenates increased enzyme activity by ~20% in a time‐ and concentration‐dependent manner. Moreover, incubation of the catalytic subunit of PKA with recombinant AAAD increased activity by ~70%. A direct phosphorylation of AAAD protein by PKA might underlie the cyclic AMP‐induced rapid and transient activation of AAAD in vivo.
Overexpression of Cu,Zn Superoxide Dismutase Attenuates Oxidative Inhibition of Astrocyte Glutamate Uptake
Authors: Chen Yongmei , Ying Weihai , Simma Vannita , Chen Yiren , Copin Jean‐Christophe , Chan Pak H. , Swanson Raymond A.
Published: March 2003

Abstract: Glutamate neurotoxicity in brain is normally prevented by rapid uptake of glutamate by astrocytes. Increased expression of Cu,Zn superoxide dismutase (SOD1) can increase resistance to cerebral ischemia and other oxidative insults, but the cellular mechanisms by which this occurs are not well established. Here we examine whether increased SOD1 expression can attenuate inhibition of astrocyte glutamate uptake by reactive oxygen species. Primary cortical astrocyte cultures were prepared from transgenic mice that overexpress human SOD1 and from nontransgenic littermate controls. Glutamate uptake was assessed after exposure of these cultures to xanthine oxidase plus hypoxanthine, an extracellular superoxide generating system, or to menadione, which generates superoxide in the cytosol. These treatments produced dose‐dependent reductions in astrocyte glutamate uptake, and the reductions were significantly attenuated in the SOD1 transgenic astrocytes. A specific effect of reactive oxygen species on glutamate transporters was suggested by the much smaller inhibitory effects of xanthine oxidase/hypoxanthine and menadione on GABA uptake than on glutamate uptake. These findings suggest that the cerebroprotective effects of increased SOD1 expression during cerebral ischemia‐reperfusion could be mediated in part by astrocyte glutamate transport.
Chronic Ethanol Exposure Attenuates the Anti‐Apoptotic Effect of NMDA in Cerebellar Granule Neurons
Authors: Bhave Sanjiv V. , Snell Lawrence D. , Tabakoff Boris , Hoffman Paula L.
Published: March 2003

Abstract: Ethanol, added to primary cultures of cerebellar granule

neurons simultaneously with NMDA, was previously shown to inhibit the

anti‐apoptotic effect of NMDA. The in vitro anti‐apoptotic effect of NMDA is

believed to mimic in vivo protection against apoptosis afforded by innervation

of developing cerebellar granule neurons by glutamatergic mossy fibers.

Therefore, the results suggested that the presence of ethanol in the brain at

a critical period of development would promote apoptosis. In the present

studies, we examined the effect of chronic ethanol exposure on the

anti‐apoptotic action of NMDA in cerebellar granule neurons. The neurons were

treated with ethanol in vitro for 1‐3 days in the absence of NMDA. Even after

ethanol was removed from the culture medium, as ascertained by gas

chromatography, the protective effect of added NMDA was significantly

attenuated. The decreased anti‐apoptotic effect of NMDA was associated with a

change in the properties of the NMDA receptor, as indicated by a decrease in

ligand binding, decreased expression of NMDA receptor subunit proteins, and

decreased functional responses including stimulation of increases in

intracellular Ca2+ and induction of brain‐derived neurotrophic factor expression. The latter effect may directly underlie the attenuated protective effect of NMDA in these neurons. The results suggest that ethanol exposure during development can have long‐lasting effects on neuronal survival. The change in the NMDA receptor caused by chronic ethanol treatment may contribute to the loss of cerebellar granule neurons that is observed in animals and humans exposed to ethanol during gestation.
Differential Expression of D1A and D1B Dopamine Receptor mRNAs in the Developing Avian Retina
Authors: Soares Heline Costa , De Melo Reis Ricardo Augusto , De Mello Fernando Garcia , Ventura Ana Lucia Marques , Kurtenbach Eleonora
Published: March 2003

Abstract: In the chick retina, the D1 dopaminergic system

differentiates very early, as shown by receptor‐mediated increases in

intracellular cyclic AMP concentration and the presence of

[3H]SCH23390‐specific binding sites. Here, we characterized, by

RT‐PCR, the expression of defined D1 receptor subtypes D1A,

D1B, and D1D during the development of the chick retina.

Total RNA was extracted from retinas of 6‐day‐old embryos (E6) to 1‐day‐old

hatched chickens and reverse‐transcribed. The resulting cDNA was amplified

using D1A‐, D1B‐, or D1D‐specific primers,

and the PCR‐amplified products were analyzed by electrophoresis. The fragment

corresponding to D1A receptor was detected in developing retina as

early as E7, whereas the fragment corresponding to D1B was observed

starting around E10. No PCR product corresponding to D1D was

observed in the retina, although it was detected in chick brain. As

synaptogenesis in chick retina begins after E11 and [3H]SCH 23390

D1 binding sites increase after this stage, the present results show that

expression of D1B receptor increases during synaptogenesis, whereas

D1A is the receptor subtype associated with the D1‐like actions of dopamine early in retina development.
Overexpression of HuD, but Not of Its Truncated Form HuD I+II, Promotes GAP‐43 Gene Expression and Neurite Outgrowth in PC12 Cells in the Absence of Nerve Growth Factor
Authors: Anderson Kim D. , Morin Melissa A. , Beckel‐Mitchener Andrea , Mobarak Charlotte D. , Neve Rachael L. , Furneaux Henry M. , Burry Richard , Perrone‐Bizzozero Nora I.
Published: March 2003

Abstract: We have previously shown that the RNA‐binding protein HuD binds to a regulatory element in the growth‐associated protein (GAP)‐43 mRNA and that this interaction involves its first two RNA recognition motifs (RRMs). In this study, we investigated the functional significance of this interaction by overexpression of human HuD protein (pcHuD) or its truncated form lacking the third RRM (pcHuD I+II) in PC12 cells. Morphological analysis revealed that pcHuD cells extended short neurites containing GAP‐43‐positive growth cones in the absence of nerve growth factor (NGF). These processes also contained tubulin and F‐actin filaments but were not stained with antibodies against neurofilament M protein. In correlation with this phenotype, pcHuD cells contained higher levels of GAP‐43 without changes in levels of other NGF‐induced proteins, such as SNAP‐25 and tau. In mRNA decay studies, HuD stabilized the GAP‐43 mRNA, whereas HuD I+II did not have any effect either on GAP‐43 mRNA stability or on the levels of GAP‐43 protein. Likewise, pcHuD I+II cells showed no spontaneous neurite outgrowth and deficient outgrowth in response to NGF. Our results indicate that HuD is sufficient to increase GAP‐43 gene expression and neurite outgrowth in the absence of NGF and that the third RRM in the protein is critical for this function.
Coupling of Canine Serotonin 5‐HT1B and 5‐HT1D Receptor Subtypes to the Formation of Inositol Phosphates by Dual Interactions with Endogenous Gi/o and Recombinant Gα15 Proteins
Authors: Wurch Thierry , Pauwels Petrus J.
Published: March 2003

Abstract: Molecular cloning and expression of canine (ca) serotonin

5‐HT1B and ca 5‐HT1D receptor subtypes showed that

besides the lower binding affinity of ketanserin for the ca 5‐HT1D

receptor, the ligand binding profiles were similar to their human homologues.

Site‐directed mutagenesis studies suggest that a Gln189 residue in

the second extracellular loop of the ca 5‐HT1D receptor may

partially account for the lower binding affinity of ketanserin. The coupling

of ca 5‐HT1B and ca 5‐HT1D receptor subtypes to the

phospholipase C pathway was analyzed by measuring stimulation of inositol

phosphate formation in COS‐7 cells. Zolmitriptan potently stimulated

(EC50 = 4.9 nM) the inositol phosphate formation at ca

5‐HT1D receptors in a fully pertussis toxin (PTX)‐dependent manner,

whereas only a weak PTX‐resistant inositol phosphate response (26‐29% at 10

μM zolmitriptan) could be detected for the ca 5‐HT1B

receptor at a similar expression level. In contrast, both ca 5‐HT1B

and ca 5‐HT1D receptor subtypes yielded a similar maximal magnitude

of inositol phosphate formation (300‐340% at 10 μM zolmitriptan)

upon co‐expression with a mouse (m) Gα15 protein. PTX

treatment and co‐expression with a β‐adrenergic receptor kinase

C‐terminal polypeptide partially (20‐46%) abolished the m

Gα15 protein‐dependent ca 5‐HT1B and ca

5‐HT1D receptor‐mediated stimulation of inositol phosphate

formation. This study suggests both 5‐HT receptor subtypes can activate

βγ subunits of endogenous Gi/o proteins besides their

coupling to recombinant m Gα15 protein.
Prevention of β‐Amyloid Neurotoxicity by Blockade of the Ubiquitin—Proteasome Proteolytic Pathway
Authors: Favit Antonella , Grimaldi Maurizio , Alkon Daniel L.
Published: March 2003

Abstract: In many neurodegenerative disorders, such as Alzheimer's disease, inclusions containing ubiquitinated proteins have been found in the brain, suggesting a pathophysiological role for ubiquitin‐mediated proteasomal degradation of neuronal proteins. Here we show for the first time that the β‐amyloid fragment 1‐40, which in micromolar levels causes the death of cortical neurons, also induces the ubiquitination of several neuronal proteins. Prevention of ubiquitination and inhibition of proteasome activity block the neurotoxic effect of β‐amyloid. These data suggest that β‐amyloid neurotoxicity may cause toxicity through the activation of protein degradation via the ubiquitin—proteasome pathway. These findings suggest possible new pharmacological targets for the prophylaxis and/or treatment of Alzheimer's disease and possibly for other related neurodegenerative disorders.
Regulators of G Protein Signaling
Authors: Burchett Scott A.
Published: March 2003

Abstract: Members of the newly discovered regulator of G protein signaling (RGS) families of proteins have a common RGS domain. This RGS domain is necessary for conferring upon RGS proteins the capacity to regulate negatively a variety of Gα protein subunits. However, RGS proteins are more than simply negative regulators of signaling. RGS proteins can function as effector antagonists, and recent evidence suggests that RGS proteins can have positive effects on signaling as well. Many RGS proteins possess additional C‐ and N‐terminal modular protein‐binding domains and motifs. The presence of these additional modules within the RGS proteins provides for multiple novel regulatory interactions performed by these molecules. These regions are involved in conferring regulatory selectivity to specific Gα‐coupled signaling pathways, enhancing the efficacy of the RGS domain, and the translocation or targeting of RGS proteins to intracellular membranes. In other instances, these domains are involved in cross‐talk between different Gα‐coupled signaling pathways and, in some cases, likely serve to integrate small GTPases with these G protein signaling pathways. This review discusses these C‐ and N‐terminal domains and their roles in the biology of the brain‐enriched RGS proteins. Methods that can be used to investigate the function of these domains are also discussed.
Characterisation of Human Adenylyl Cyclase IX Reveals Inhibition by Ca2+/Calcineurin and Differential mRNA Plyadenylation
Authors: Paterson Janice M. , Smith Susan M. , Simpson James , Grace O. Cathal , Sosunov Alexander A. , Bell Jeanne E. , Antoni Ferenc A.
Published: March 2003

Abstract: The functional diversity of adenylyl cyclases provides for
different modes of cyclic AMP signalling in mammals. This study reports the
cloning and functional characterisation of a cDNA encoding human adenylyl
cyclase IX (ACIX). The data show that human ACIX is a
Ca2+/calcineurin‐inhibited adenylyl cyclase prominently expressed
in vital organs, including brain, heart, and pancreas. ACIX mRNA was detected
in several brain regions, including neocortex, hippocampus, striatum, and
cerebellum. By in situ hybridisation, ACIX mRNA was localised to pyramidal and
granule cells of the hippocampus, indicating that it is expressed
predominantly in nerve cells. Further analysis of ACIX mRNA expression
revealed two major forms of ACIX mRNA that arose through tissue‐specific
differential mRNA polyadenylation. Taken together, the data show that (a)
human ACIX is under inhibitory control by Ca2+ through calcineurin, (b) ACIX may be involved in higher brain functions, and (c) post‐transcriptional regulation of ACIX gene expression is a species‐specific control mechanism that may enhance the versatility of cyclic AMP signalling in humans.
The Effects of Methamphetamine on Serotonin Transporter Activity
Authors: Haughey Heather M. , Fleckenstein Annette E. , Metzger Ryan R. , Hanson Glen R.
Published: March 2003

Abstract: Multiple administrations of methamphetamine (METH) rapidly
decreased serotonin (5HT) transporter (SERT) function in rat striatum and
hippocampus. The purpose of this study was to identify the mechanisms/ factors
contributing to this METH‐induced decrease in SERT function. Multiple
high‐dose METH injections rapidly decreased 5HT uptake without altering
binding of the 5HT transporter ligand paroxetine. Hyperthermia contributed to
this deficit in transporter function in striatum and hippocampus, as
prevention of METH‐induced hyperthermia attenuated this decrease. A role for
dopamine (DA) was suggested by findings that pretreatment with the tyrosine
hydroxylase inhibitor α‐methyl‐p‐tyrosine, the D1 antagonist SCH‐23390, or the D2 antagonist eticlopride attenuated the METH‐induced decrease in striatal, but not hippocampal, SERT activity. These effects were independent of the ability of these DA‐antagonizing drugs to prevent METH‐induced hyperthermia. These results suggest that DA contributes to the decrease in SERT function caused by multiple METH injections in the striatum, but not hippocampus, and that hyperthermia facilitates these deficits in SERT function in both brain regions. In contrast, the response of SERT to a single administration of METH was DA and hyperthermia independent. These findings suggest that the mechanisms/ factors involved in decreasing SERT activity after a single administration of METH are distinct from that caused by multiple administrations.
Constitutive Phosphorylation of the Vesicular Inhibitory Amino Acid Transporter in Rat Central Nervous System
Authors: Bedet Cécile , Isambert Marie‐Françoise , Henry Jean‐Pierre , Gasnier Bruno
Published: March 2003

Abstract:γ‐Aminobutyric acid (GABA) and glycine are stored
into synaptic vesicles by a recently identified vesicular
inhibitory amino acid transporter
[VIAAT, also called vesicular GABA
transporter (VGAT)]. Immunoblotting analysis revealed that rat brain VIAAT migrated as a doublet during sodium dodecyl sulfate—polyacrylamide gel electrophoresis, with a predominant slower band in all areas examined except olfactory bulb and retina. The slower band corresponded to a phosphorylated form of VIAAT as it was converted to the faster one by treating brain homogenates with alkaline phosphatase or with an endogenous phosphatase identified as type 2A protein—serine/threonine phosphatase using okadaic acid. In contrast, the recombinant protein expressed in COS‐7 or PC12 cells co‐migrated with the faster band of the brain doublet and was insensitive to alkaline phosphatase. To investigate the influence of VIAAT phosphorylation on vesicular neurotransmitter loading, purified synaptic vesicles were treated with alkaline phosphatase and assayed for amino acid uptake. However, neither GABA nor glycine uptake was affected by VIAAT phosphorylation. These results indicate that VIAAT is constitutively phosphorylated on cytosolic serine or threonine residues in most, but not all, regions of the rat brain. This phosphorylation does not regulate the vesicular loading of GABA or glycine, suggesting that it is involved at other stages of the synaptic vesicle life cycle.
Structural Implications on the Interaction of Scorpion α‐like Toxins with the Sodium Channel Receptor Site Inferred from Toxin Iodination and pH‐Dependent Binding
Authors: Gilles Nicolas , Krimm Isabelle , Bouet Francoise , Froy Oren , Gurevitz Michael , Lancelin Jean‐Marc , Gordon Dalia
Published: March 2003

Abstract: The α‐like toxin from the venom of the scorpion
Leiurus quinquestriatus hebraeus (Lqh III) binds with high affinity
to receptor site 3 on insect sodium channels but does not bind to rat brain
synaptosomes. The binding affinity of Lqh III to cockroach neuronal membranes
was fivefold higher at pH 6.5 than at pH 7.5. This correlated with an increase
in the electropositive charge on the toxin surface resulting from protonation
of its four histidines. Radioiodination of Tyr14 of Lqh III
abolished its binding to locust but not cockroach sodium channels, whereas the
noniodinated toxin bound equally well to both neuronal preparations.
Radioiodination of Tyr10 or Tyr21 of the structurally
similar α‐toxin from L. quinquestriatus hebraeus
(LqhαIT), as well as their substitution by phenylalanine, had only minor
effects on binding to cockroach neuronal membranes. However, substitution of
Tyr21, but not Tyr14, by leucine decreased the binding
affinity of LqhαIT ∼87‐fold. Thus, Tyr14 is involved in
the bioactivity of Lqh III to locust receptor site 3 and is not crucial for
the binding of LqhαIT to this site. In turn, the aromatic ring of
Tyr21 takes part in the bioactivity of LqhαIT to insects. These results highlight subtle architectural variations between locust and cockroach receptor site 3, in addition to previous results demonstrating the competence of Lqh III to differentiate between insect and mammalian sodium channel subtypes.
Effect of Chronic Psychogenic Stress Exposure on Enkephalin Neuronal Activity and Expression in the Rat Hypothalamic Paraventricular Nucleus
Authors: Dumont E. C. , Kinkead R. , Trottier J.‐F. , Gosselin I. , Drolet G.
Published: March 2003

Abstract: This study tested the hypothesis that the activation pattern of enkephalinergic (ENKergic) neurons within the paraventricular nucleus of the hypothalamus (PVH) in response to psychogenic stress is identical whether in response to repeated exposure to the same stress (homotypic; immobilization) or to a novel stress (heterotypic; air jet puff). Rats were assigned to either acute or chronic immobilization stress paradigms (90 min/day for 1 or 10 days, respectively). The chronic group was then subjected to an additional 90‐min session of either heterotypic or homotypic stress. A single 90‐min stress session (immobilization or air jet) increased PVH‐ENK heteronuclear (hn) RNA expression. In chronically stressed rats, exposure to an additional stress session (whether homotypic or heterotypic) continued to stimulate ENK hnRNA expression. Acute immobilization caused a marked increase in the numbers of Fos‐immunoreactive and Fos‐ENK double‐labeled cells in the dorsal and ventral medial parvicellular, and lateral parvicellular subdivisions of the PVH. Chronic immobilization caused an attenuated Fos response (∼ 66%) to subsequent immobilization. In contrast, chronic immobilization did not impair ENKergic neuron activation within the PVH following homotypic or heterotypic stress. These results indicate that within the PVH, chronic psychogenic stress markedly attenuates the Fos response, whereas ENKergic neurons resist habituation, principally within the ventral neuroendocrine portion of the nucleus. This suggests an increase in ENK effect during chronic stress exposure. Homotypic (immobilization) and heterotypic (air jet) psychogenic stressors produce similar responses, including Fos, ENK‐Fos, and ENK hnRNA, within each subdivision of the PVH, suggesting similar processing for painless neurogenic stimuli.
(+)‐ and (‐)‐cis‐2‐Aminomethylcyclopropanecarboxylic Acids Show Opposite Pharmacology at Recombinant ρ1 and ρ2 GABAC Receptors
Authors: Duke Rujee K. , Chebib Mary , Balcar Vladimir J. , Allan Robin D. , Mewett Kenneth N. , Johnston Graham A. R.
Published: March 2003

Abstract: The effects of the enantiomers of (±)‐CAMP and(±)‐TAMP [(±)‐cis‐ and(±)‐trans‐2‐aminomethylcyclopropanecarboxylic acids,respectively], which are cyclopropane analogues of GABA, were tested onGABAA and GABAC receptors expressed in Xenopuslaevis oocytes using two‐electrode voltage clamp methods. (+)‐CAMP wasfound to be a potent and full agonist at homooligomeric GABACreceptors (KD∼40 μM andImax∼100% at ρ1;KD∼17 μM and Imax∼100% at ρ2) but a very weak antagonist atα1β2γ2L GABAAreceptors. In contrast, (‐)‐CAMP was a very weak antagonist at bothα1β2γ2L GABAAreceptors and homooligomeric GABAC receptors (IC50∼900 μM at ρ1 and ∼400 μM atρ2). Furthermore, (+)‐CAMP appears to be a superior agonist tothe widely used GABAC receptor partial agonistcis‐4‐aminocrotonic acid (KD∼74μM and Imax∼78% at ρ1;KD∼70 μM and Imax∼82% at ρ2). (‐)‐TAMP was the most potent of thecyclopropane analogues on GABAC receptors (KD∼9 μM and Imax∼40% atρ1; KD∼3 μM andImax∼50‐60% at ρ2), but it was also amoderately potent GABAA receptor partial agonist(KD∼50‐60 μM and Imax∼50% at α1β2γ2LGABAA receptors). (+)‐TAMP was a less potent partial agonist atGABAC receptors (KD∼60 μM andImax∼40% at ρ1; KD∼30 μM and Imax∼60% atρ2) and a weak partial agonist atα1β2γ2L GABAAreceptors (KD∼500 μM andImax∼50%). None of the isomers of (±)‐CAMP and(±)‐TAMP displayed any interaction with GABA transport at theconcentrations tested. Molecular modeling based on the present resultsprovided new insights into the chiral preferences for either agonism orantagonism at GABAC receptors.
Analysis of Gap Junction Assembly Using Mutated Connexins Detected in Charcot—Marie—Tooth X‐Linked Disease
Authors: Martin Patricia E. M. , Mambetisaeva Elvira T. , Archer Deborah A. , George Christopher H. , Evans W. Howard
Published: March 2003

Abstract: The assembly of gap junction intercellular communication channels was studied by analysis of the molecular basis of the dysfunction of connexin 32 mutations associated with the X‐linked form of Charcot—Marie—Tooth disease in which peripheral nervous transmission is impaired. A cell‐free translation system showed that six recombinant connexin 32 mutated proteins—four point mutations at the cytoplasmic amino terminus, one at the membrane aspect of the cytoplasmic carboxyl terminus, and a deletion in the intracellular loop—were inserted into microsomal membranes and oligomerised into connexon hemichannels with varying efficiencies. The functionality of the connexons was determined by the ability of HeLa cells expressing the respective connexin cDNAs to transfer Lucifer yellow. The intracellular trafficking properties of the mutated connexins were determined by immunocytochemistry. The results show a relationship between intracellular interruption of connexin trafficking, the efficiency of intercellular communication, and the severity of the disease phenotype. Intracellular retention was explained either by deficiencies in the ability of connexins to oligomerise or by mutational changes at two targeting motifs. The results point to dominance of two specific targeting motifs: one at the amino terminus and one at the membrane aspect of the cytoplasmically located carboxyl tail. An intracellular loop deletion of six amino acids, associated with a mild phenotype, showed partial oligomerisation and low intercellular dye transfer compared with wild‐type connexin 32. The results show that modifications in trafficking and assembly of gap junction channels emerge as a major feature of Charcot—Marie—Tooth X‐linked disease.
Accumulation of the anandamide precursor and other N‐acylethanolamine phospholipids in infant rat models of in vivo necrotic and apoptotic neuronal death
Authors: Hansen Henrik H. , Ikonomidou Chrysanthy , Bittigau Petra , Hansen Steen Honoré , Hansen Harald S.
Published: March 2003

It has been demonstrated that the endogenous cannabinoid receptor ligand, anandamide, and other N‐acylethanolamines (NAEs), accumulate during neuronal injury in vitro, a process that may be linked to the neuroprotective effects of NAEs. The crucial step for generation of NAEs is the synthesis of the corresponding precursors, N‐acylethanolamine phospholipids (NAPEs). However, it is unknown whether this key event for NAE formation is regulated differently in the context of insults causing necrotic or apoptotic neuronal death. To address this question, we monitored a range of cortical NAPE species in three infant rat models of in vivo neurodegeneration: (i) necrosis caused by intrastriatal injection of NMDA (25 nmol); (ii) apoptosis induced by systemic administration of the NMDA‐receptor antagonist (+)MK‐801 (3 × 0.5 mg/kg, i.p.); and (iii) apoptosis following focal necrosis triggered by concussive head trauma. A marked increase of all NAPE species was observed in both hemispheres 4 and 24 h after NMDA‐induced injury, with a relatively larger increase in N‐stearoyl‐containing NAPE species. Thus, the percentage of the anandamide precursor fell from 1.1 to 0.5 mol %. In contrast, administration of (+)MK‐801 did not alter cortical NAPE levels. Concussion head trauma resulted in a similar but less pronounced upregulation of NAPE levels at both 4 and 24 h as compared to NMDA injections. Increased levels of NAPE 24 h post‐trauma possibly reflect that necrosis is still ongoing at this time point. Consequently, our data suggest that excitotoxic necrotic mechanisms of neurodegeneration, as opposed to apoptotic neurodegeneration, have a profound effect on in vivo NAE precursor homeostasis.
Sex steroid regulation of glutamate decarboxylase mRNA expression in goldfish brain is sexually dimorphic
Authors: Bosma Peter T. , Blázquez Mercedes , Fraser E. Jane , Schulz Rüdiger W. , Docherty Kevin , Trudeau Vance L.
Published: March 2003

Testosterone and oestradiol can modulate GABA synthesis in sexually regressed goldfish. Here we investigated their effects on the mRNA expression of two isoforms of the GABA synthesizing enzyme glutamate decarboxylase (GAD65and GAD67, EC 4.1.1.15). Full‐length GAD clones were isolated from a goldfish cDNA library and sequenced. Goldfish GAD65encodes a polypeptide of 583 amino acid residues, which is 77% identical to human GAD65. Goldfish GAD67encodes a polypeptide of 587 amino acid residues and is 82% identical to human GAD67. Goldfish GAD65and GAD67are 63% identical. Sexually regressed male and female goldfish were implanted with solid silastic pellets containing testosterone, oestradiol or no steroid. Semiquantitative PCR analysis showed that oestradiol significantly increased GAD65mRNA expression in female hypothalamus and telencephalon, while testosterone resulted in a significant increase only in telencephalon. GAD67mRNA levels were not affected by steroids in females. In contrast, both steroids induced significant decreases of GAD65and GAD67mRNA levels in male hypothalamus, but had no effect on GAD mRNA expression in male telencephalon. Our results indicate that modulation of GAD mRNA expression is a possible mechanism for steroid action on GABA synthesis, which may have opposite effects in males and females.
Molecular cloning, localization and characterization of a 40‐kDa catecholamine‐regulated protein
Authors: Nair Venugopalan D. , Mishra Ram K.
Published: March 2003

We have previously described catecholamine‐regulated proteins of molecular masses 47, 40 and 26 kDa (CRP47/40/26). In mammals, these proteins are detected only in brain and have been implicated as playing a role in dopaminergic neurotransmission. In this report, we have cloned the cDNA encoding CRP40 from bovine brain. Analysis of the predicted amino acid sequence revealed that the CRP40 product contains an hsp70 motif and shares homology with heat‐shock protein hsp70. Immunolocalization studies using mAbs to dopamine show that it colocalizes with CRP40 in the vesicles of dopaminergic neuroblastoma SH‐SY5Y cells. The constitutive expression of CRP40 was increased by exposure to heat shock similar to inducible heat‐shock protein hsp70 in SH‐SY5Y cells. Dopamine significantly modulated the levels of CRP40, whereas, the expression of hsp70 remained unchanged upon dopamine treatment of these cells. Moreover, CRP40 is able to prevent the thermal aggregation of luciferase in vitro, similar to hsp70, suggesting that CRP40 encodes a dopamine‐inducible protein with properties similar to heat‐shock proteins. The immunofluorescence analyses show that in SH‐SY5Y cells, CRP40 translocates to the nucleus during dopamine‐induced apoptosis. These results suggest that CRP40 could play a protective role against the harmful effects of catecholamine metabolites.