bluuebunny/crossref_metadata_2025 huggingface dataset:
Brief Exposure to Neurotrophins Produces a Calcium‐Dependent Increase in Choline Acetyltransferase Activity in Cultured Rat Septal Neurons
Authors: Nonner Doris , Barrett Ellen F. , Barrett John N.
Published: March 2003

Abstract: We demonstrate that brief (30‐min) exposure of cultured
embryonic rat septal neurons to neurotrophins (NTs) increases choline
acetyltransferase (ChAT) activity by 20‐50% for all tested NTs (nerve growth
factor, brain‐derived neurotrophic factor, neurotrophin‐3, and neurotrophin‐4,
each at 100 ng/ml). The increase in ChAT activity was first detected 12 h
after NT exposure, persisted at least 48 h, and was not mediated by increased
neuronal survival or action‐potential activity. Under some conditions, the
response to brief NT exposure was as great as that produced by continuous
exposure. NT stimulation of ChAT activity was inhibited by inhibitors of p75‐
and Trk kinase‐mediated signaling, by removal of extracellular Ca2+
during the period of NT exposure, and by buffering intracellular
Ca2+ with BAPTA. Application of nerve growth factor and
brain‐derived neurotrophic factor transiently increased [Ca2+]
within a subpopulation of neurons. NT stimulation of ChAT activity was not
affected significantly by cyclic AMP agonists or antagonists. These findings
suggest that brief exposure to NTs can have a long‐lasting effect on
cholinergic transmission, and that this effect requires Ca2+, but not cyclic AMP.
Attenuation and Recovery of Evoked Overflow of Striatal Serotonin in Rats Treated with Neurotoxic Doses of Methamphetamine
Authors: Cass Wayne A.
Published: March 2003

Abstract: Repeated administration of methamphetamine to animals can lead to long‐lasting decreases in striatal monoamine content. In the present study, the effects of neurotoxic doses of methamphetamine on basal and evoked overflow of striatal serotonin and of its primary metabolite 5‐hydroxyindoleacetic acid were examined in awake rats using in vivo microdialysis. Male Fischer‐344 rats were administered methamphetamine (5 mg/kg, s.c.) or saline four times in 1 day at 2‐h intervals. Microdialysis studies were carried out 1 week, 1 month, and 6 months later. At 1 week posttreatment there were significant decreases in potassium‐ and amphetamine‐evoked overflow of serotonin in the striatum of the methamphetamine‐treated animals. Basal extracellular levels of 5‐hydroxyindoleacetic acid but not of serotonin were also decreased. Evoked overflow of serotonin recovered by 1 month, and extracellular levels of 5‐hydroxyindoleacetic acid had recovered by 6 months. Tissue levels of serotonin and 5‐hydroxyindoleacetic acid were decreased at 1 week posttreatment but back to control levels by 1 month after treatment. These results indicate that presynaptic serotonergic functioning is attenuated in the striatum of rats treated 1 week earlier with neurotoxic doses of methamphetamine. However, in the model used, the changes are transient, and recovery can occur within 1‐6 months posttreatment.
Regulation of α3 Nicotinic Acetylcholine Receptor Subunit mRNA Levels by Nerve Growth Factor and Cyclic AMP in PC12 Cells
Authors: Nakayama Hitoshi , Ueno Satoshi , Ikeuchi Toshihiko , Hatanaka Hiroshi
Published: March 2003

Abstract: To investigate the effects of nerve growth factor (NGF)
and cyclic AMP (cAMP) on the level of the nicotinic acetylcholine receptor
subunit α3 mRNA, we used PC12h cells, PC12 cells expressing
dominant‐negative Ras protein, and the parental PC12 cells. PC12h cells have
NGF‐responsive tyrosine hydroxylase activity. Expression of dominant‐negative
Ras protein prevents the signaling through the Ras‐mitogen‐activated protein
kinase cascade. The morphological changes of the parental PC12 cells in
response to NGF and 8‐(4‐chlorophenylthio)adenosine 3′,5′‐cyclic
monophosphate (CPTcAMP), a cell‐penetrating cAMP analogue, were similar to
those of PC12h cells. NGF up‐regulated the α3 mRNA level in PC12h cells
and down‐regulated the α3 mRNA level in the parental PC12 cells.
Expression of dominant‐negative Ras protein and an inhibitor of
mitogen‐activated protein kinase kinase inhibited the effects of NGF on
α3 mRNA level. CPTcAMP down‐regulated the α3 mRNA level in all
three PC12 cell lines. An inhibitor of protein kinase A inhibited the
CPTcAMP‐induced down‐regulation of α3 mRNA. The α3 mRNA
down‐regulation required prolonged treatment with CPTcAMP even after cAMP
response element binding protein phosphorylation was decreased. Membrane
depolarization with high K+ had no effect on the α3 mRNA level in PC12h cells. Based on these results, we propose that at least two unknown effectors regulate α3 mRNA levels in PC12 cells.
Sodium Nitroprusside Prevents Chemical Hypoxia‐Induced Cell Death Through Iron Ions Stimulating the Activity of the Na+‐Ca2+ Exchanger in C6 Glioma Cells
Authors: Amoroso Salvatore , Tortiglione Anna , Secondo Agnese , Catalano Annalisa , Montagnani Stefania , Di Renzo Gianfranco , Annunziato Lucio
Published: March 2003

Abstract: In C6 glioma cells exposed to chemical hypoxia, an
increase of extracellular lactate dehydrogenase (LDH) activity, cell death,
and intracellular Ca2+ concentration
([Ca2+]i) occurred. Sodium nitroprusside (SNP), a nitric
oxide donor and an iron‐containing molecule, reduced chemical hypoxia‐induced
LDH release and cell death. These effects were counteracted by bepridil and by
5‐(N‐4‐chlorobenzyl)‐2′,4′‐dimethylbenzamil (CB‐DMB), two
specific inhibitors of the Na+‐Ca2+ exchanger. SNP also
increased the activity of the Na+‐Ca2+ exchanger as a
Na+ efflux pathway, stimulated by Na+‐free conditions
and evaluated by monitoring [Ca2+]i in single cells. In
addition, SNP produced a further increase of chemical hypoxia‐elicited
[Ca2+]i elevation, and this effect was blocked by
bepridil. Chemical hypoxiaevoked cell death and LDH release were counteracted
by the ferricyanide moiety of the SNP molecule,
K3Fe(CN)6, and by ferric chloride (FeCl3),
and this effect was counteracted by CB‐DMB. In addition, the iron ion chelator
deferoxamine reversed the protective effect exerted by SNP on cell injury.
Collectively, these findings suggest that the protective effect of SNP on C6
glioma cells exposed to chemical hypoxia is due to the activation of the
Na+‐Ca2+ exchanger operating as a Na+
efflux‐Ca2+ influx pathway induced by iron present in the SNP molecule.
Norepinephrine‐Stimulated Increase in Na+,K+‐ATPaseActivity in the Rat Brain Is Mediated Throughα1A‐Adrenoceptor Possibly by Dephosphorylation of the Enzyme
Authors: Mallick Birendra Nath , Adya H. V. Anupama , Faisal Mohammad
Published: March 2003

Abstract: Rapid eye movement sleep deprivation is reported to
increase Na+,K+‐ATPase activity. This increase was shown
earlier to be stimulated by norepinephrine acting on
α1‐adrenoceptor. The involvement of a subtype of
α1‐adrenoceptor and the possible molecular mechanism of
action of norepinephrine in increasing the enzyme activity were investigated
using receptor agonists and antagonists, as well as stimulants and blockers of
signal transduction pathway. It was observed that incubation of the homogenate
with cyclic AMP, forskolin, A23187 (a calcium ionophore), or calmodulin alone
did not stimulate the Na+,K+‐ATPase activity. However,
although the spontaneous activity of the Na+,K+‐ATPase
was not affected by prazosin, WB4101, heparin, W13, or cyclosporin A alone,
each of them could prevent the norepinephrine‐stimulated increase in the
enzyme activity. Based on these results and our previous findings, it is
proposed that norepinephrine acted on α1A‐adrenoceptor and
increased intracellular calcium, which in the presence of calmodulin activated
a calmodulin‐dependent phosphatase, calcineurin. This calcineurin possibly
dephosphorylated Na+,K+‐ATPase and increased its activity. The physiological significance especially in relation to rapid eye movement sleep deprivation is discussed.
Protein Kinase C‐α and ‐ε Down‐Regulate Cell Surface Sodium Channels via Differential Mechanisms in Adrenal Chromaffin Cells
Authors: Yanagita Toshihiko , Kobayashi Hideyuki , Yamamoto Ryuichi , Kataoka Hiroaki , Yokoo Hiroki , Shiraishi Seiji , Minami Shin‐ichi , Koono Masashi , Wada Akihiko
Published: March 2003

Abstract: In cultured bovine adrenal chromaffin cells, our
[3H]saxitoxin ([3H]STX) binding, immunoblot, and
northern blot analyses specified protein kinase C (PKC) isoform‐specific
posttranscriptional and posttranslational mechanisms that direct
down‐regulation of cell surface Na channels. Immunoblot analysis showed that
among 11 PKC isoforms, adrenal chromaffin cells contained only conventional
(c)PKC‐α, novel (n)PKC‐ε, and atypical (a)PKC‐ζ. Treatment of
adrenal chromaffin cells with 100 nM
12‐O‐tetradecanoylphorbol 13‐acetate (TPA) or 100 nM phorbol
12,13‐dibutyrate (PDBu) caused a rapid (< 15 min) and sustained (> 15 h)
translocation of PKC‐α and ‐ε (but not ‐ζ) from cytosol to
membranes, whereas a biologically inactive 4α‐TPA had no effect.
Thymeleatoxin (TMX), an activator of cPKC, produced similar membrane
association of only PKC‐α at 100 nM, with the potency of TMX
being comparable with those of TPA and PDBu. Treatment with either 100
nM TPA or 100 nM TMX reduced cell surface [3H]STX
binding to a comparable extent at 3, 6, and 12 h, whereas TPA lowered the
binding to a greater extent than TMX at 15, 18, and 24 h; at 15 h,
Gö6976, a specific inhibitor of cPKC, completely
blocked TMX‐induced decrease of [3H]STX binding while preventing by
merely 57% TPA‐induced decrease of [3H]STX binding. Treatment with
100 nM TPA lowered the Na channel α‐subunit mRNA level between
3 and 12 h, with its maximum 52% fall at 6 h, and it was accompanied by a
subsequent 61% rise of the β1‐subunit mRNA level at 24 h.
Gö6976 failed to prevent TPA‐induced reduction
of the α‐subunit mRNA level; TMX did not change the α‐and
β1‐subunit mRNA levels throughout the 24‐h treatment.
Brefeldin A, an inhibitor of vesicular exit from the trans‐Golgi
network, augmented TPA‐ and TMX‐induced decrease of [3H]STX binding at 1 and 3 h. Our previous and present studies suggest that PKC down‐regulates cell surface Na channels without altering the allosteric gating of Na channels via PKC isoform‐specific mechanisms; cPKC‐α promotes Na channel internalization, whereas nPKC‐ε decreases the α‐subunit mRNA level by shortening the half‐life of α‐subunit mRNA without changing its gene transcription.
A Role for the Small Molecular Weight GTPases, Rho and Cdc42, in Muscarinic Receptor Signaling to Focal Adhesion Kinase
Authors: Linseman Daniel A. , Hofmann Fred , Fisher Stephen K.
Published: March 2003

Abstract: An enhanced tyrosine phosphorylation of focal adhesion kinase (FAK) is elicited during neuronal growth cone remodeling and requires the maintenance of agonist‐sensitive pools of phosphatidylinositol 4,5‐bisphosphate (PIP2). Rho family GTPases are putative regulators of both PIP2 synthesis and growth cone remodeling, including neurite outgrowth elicited by muscarinic cholinergic receptor (mAChR) stimulation. In this study, we investigated the interrelationships among Rho family GTPases, PIP2 synthesis, and mAChR signaling to FAK in SH‐SY5Y neuroblastoma cells. Preincubation with Clostridium difficile toxin B (Tox B), an inhibitor of Rho, Rac, and Cdc42, attenuated mAChR‐stimulated FAK and paxillin tyrosine phosphorylation and lysophosphatidic acid (LPA)‐induced FAK phosphorylation to a similar extent (75% decreases at 200 pg/ml Tox B) but did not affect mitogen‐activated protein kinase activation elicited by either phorbol ester or an mAChR agonist. In contrast, preincubation with selective inhibitors of either Rho (C3 exoenzyme) or Rho kinase (HA‐1077) resulted in 80‐90% reductions in LPA‐induced FAK phosphorylation but only 40‐50% decreases in mAChR‐stimulated phosphorylation. Moreover, mAChR‐mediated FAK phosphorylation was significantly attenuated in cells scrape‐loaded with dominant‐negative N17Cdc42 but not N17Rac1. Tox B had little or no effect on agonist‐sensitive pools of PIP2 but inhibited mAChR‐driven actin cytoskeletal remodelling. The results suggest that the Rho family GTPases, Rho and Cdc42, link mAChR stimulation to increases in FAK phosphorylation independently of effects on PIP2 synthesis.
Analysis of VMAT2 Binding After Methamphetamine or MPTP Treatment
Authors: Hogan Kelly A. , Staal Roland G. W. , Sonsalla Patricia K.
Published: March 2003

Abstract: [3H]Dihydrotetrabenazine ([3H]DTBZ), a specific ligand for the vesicular monoamine transporter (VMAT2), has been used to characterize the integrity of monoaminergic nerve terminals in experimental animals and humans. The purpose of the present studies was to compare the loss of VMAT2 binding with the loss of other neurochemical markers of the dopamine (DA) nerve terminals in mice treated with neurotoxic doses of methamphetamine (METH) or MPTP. Profound decreases (≥70%) in DA content, tyrosine hydroxylase activity, and [3H]carbomethoxy‐3‐(4‐fluorophenyl)tropane binding to the DA transporter were observed in striatal homogenates at both 1 and 6 days after exposure to the neurotoxins. It is surprising that no significant loss of [3H]DTBZ binding in the homogenates was observed at 1 day after exposure, although a significant loss (‐50%) was apparent 6 days later. However, in isolated vesicle preparations, [3H]DTBZ binding and active [3H]DA uptake were markedly reduced (>70%) at 1 day. These observations indicate that vesicle function is compromised at an early time point after exposure to neurotoxic insult. Furthermore, the changes in [3H]DTBZ binding in homogenates may not be a sensitive indicator of early damage to synaptic vesicles, although homogenate binding reliably identifies a loss of VMAT2 at later times.
Methamphetamine Rapidly Decreases Vesicular Dopamine Uptake
Authors: Brown Jeffrey M. , Hanson Glen R. , Fleckenstein Annette E.
Published: March 2003

Abstract: Vesicular sequestration is important in the regulation of cytoplasmic concentrations of monoamines such as dopamine. Moreover, recent evidence suggests that increases in cytoplasmic dopamine levels, perhaps attributable to changes in vesicular monoamine transporter function, contribute to methamphetamine‐induced dopaminergic deficits. Hence, we examined whether striatal vesicular uptake is altered following methamphetamine treatment. Multiple administrations of methamphetamine rapidly (within 1 h) decreased vesicular dopamine uptake and dihydrotetrabenazine binding, an effect that (a) persisted at least 24 h, (b) was associated with dopamine and not serotonin neurons, and (c) was unrelated to residual drug introduced by the original methamphetamine treatment. These data suggest that methamphetamine rapidly decreases vesicular monoamine transporter function in dopaminergic neurons, a phenomenon that may be associated with the long‐term damage caused by this stimulant.
The Salt‐Inducible Kinase, SIK, Is Induced by Depolarization in Brain
Authors: Feldman Jonathan D. , Vician Linda , Crispino Marianna , Hoe Warren , Baudry Michel , Herschman Harvey R.
Published: March 2003

Abstract: Membrane depolarization of neurons is thought to lead to changes in gene expression that modulate neuronal plasticity. We used representational difference analysis to identify a group of cDNAs that are induced by membrane depolarization or by forskolin, but not by neurotrophins or growth factors, in PC12 pheochromocytoma cells. One of these genes, SIK (salt‐inducible kinase), is a member of the sucrose‐nonfermenting 1 protein kinase/AMP‐activated protein kinase protein kinase family that was also recently identified from the adrenal gland of rats treated with high‐salt diets. SIK mRNA is induced up to eightfold in specific regions of the hippocampus and cortex in rats, following systemic kainic acid administration and seizure induction.
Unusual Regulation of Cyclin D1 and Cyclin‐Dependent Kinases cdk2 and cdk4 During In Vivo Mitotic Stimulation of Olfactory Neuron Progenitors in Adult Mouse
Authors: Kastner A. , Moyse E. , Bauer S. , Jourdan F. , Brun G.
Published: March 2003

Abstract: The molecular mechanisms underlying cell cycle control in neuronal progenitors have been investigated with adult mouse olfactory epithelium as a model system. Odorreceptive neurons of mammalian olfactory epithelium are short‐lived and renewed in the adult by mitotic division of intrinsic neuronal progenitors. Ablation of the synaptic target, olfactory bulb, induces sequentially extensive apoptosis of sensory neurons and then stimulation of progenitor proliferation, peaking at 36 h and 4 days, respectively, postlesion. Known molecular effectors of G1 phase entry have been assessed on protein extracts of olfactory organs sampled at various postbulbectomy times in adult mice. The decay of βIII‐tubulin and olfactory marker protein levels and the rise of proliferating cell nuclear antigen (PCNA) levels, starting 1 and 3 days, respectively, postlesion, provided the kinetic frame of neuronal dynamics. Cyclin D1, cyclin E, and cyclin‐dependent kinase cdk2 levels, low in olfactory organ of intact mice, increased 3 days after bulbectomy in parallel with PCNA levels; cdk4 content was initially high and unaffected by lesioning. Western blots of the known cdk inhibitors revealed proliferation‐related decreases of p18, p21, and p27 from high expression in intact organs. Immunoprecipitation of cdk2 and cdk4 fractions of protein extracts at 4 days postlesion (mitotic reaction peak) versus control, followed by cyclin D1 immunoblotting, and vice versa, revealed that levels of both cyclin D1/cdk2 and cyclin D1/cdk4 complexes, as well as their kinase activities, were dramatically increased after lesion. In vivo proliferation of olfactory neuronal lineage cells thus involves functional binding of cyclin D1 with cdk2 and cdk4, with differential activation mechanisms for cdk2 and cdk4. In addition, the RT‐PCR‐detected cyclin D1 mRNA level remained unaffected after bulbectomy, which indicated that the cyclin D1 rise should involve posttranscriptional mechanisms in this in vivo neuronal system. These observations are discussed, along with their relevance to cell cycle control and to olfactory neuron dynamics.
Enhancement of Diphtheria Toxin Potency by Replacement of the Receptor Binding Domain with Tetanus Toxin C‐Fragment
Authors: Francis Jonathan W. , Brown Robert H. , Figueiredo Dayse , Remington Mary P. , Castillo Orlando , Schwarzschild Michael A. , Fishman Paul S. , Murphy John R. , VanderSpek Johanna C.
Published: March 2003

Abstract: This study describes the expression, purification, and characterization of a recombinant fusion toxin, DAB389TTC, composed of the catalytic and membrane translocation domains of diphtheria toxin (DAB389) linked to the receptor binding fragment of tetanus toxin (C‐fragment). As determined by its ability to inhibit cellular protein synthesis in primary neuron cultures, DAB389TTC was ∼ 1,000‐fold more cytotoxic than native diphtheria toxin or the previously described fusion toxin, DAB389MSH. The cytotoxic effect of DAB389TTC on cultured cells was specific toward neuronal‐type cells and was blocked by coincubation of the chimeric toxin with tetanus antitoxin. The toxicity of DAB389TTC, like that of diphtheria toxin, was dependent on passage through an acidic compartment and ADP‐ribosyltransferase activity of the DAB389 catalytic fragment. These results suggest that a catalytically inactive form of DAB389TTC may be useful as a nonviral vehicle to deliver exogenous proteins to the cytosolic compartment of neurons.
Electrophysiological Evidence for the Coexistence of α1 and α6 Subunits in a Single Functional GABAA Receptor
Authors: Sigel Erwin , Baur Roland
Published: March 2003

Abstract: The subunit combinations α1β2γ2, α6β2γ2, and α1α6β2γ2 of the GABAA receptor were functionally expressed in Xenopus oocytes. The properties of the resulting ion currents were characterized by using electrophysiological techniques. The concentration‐response curve of the channel agonist GABA for α1α6β2γ2 showed a single apparent component characterized by an EC50 of 107 ± 26 μM (n = 4). It was different from the one for α1β2γ2, which had an EC50 of 41 ± 9 μM (n = 4), that for α6β2γ2, with an EC50 of 6.7 ± 1.9 μM (n = 5), and those for α1β2 and α1α6β2. There was no appreciable functional expression of α6β2. Allosteric responses of α1α6β2γ2 to diazepam were intermediate to those of α1β2γ2 and α6β2γ2, and allosteric responses to flumazenil were comparable to the ones for α1β2γ2. The inhibition by furosemide of the currents elicited by GABA in α1α6β2γ2 [IC50 = 298 ± 116 μM (n = 7), assuming only one component] was not identical with inhibition of α6β2γ2 (IC50 = 38 ± 2 μM, n = 4), α1β2γ2 (IC50 = 5,610 ± 910 μM, n = 5), or a mixture of these components (assuming two components). These findings indicate unambiguously the formation of functional GABAA receptors containing two different α subunits, α1 and α6, with properties different from those of α1β2γ2 and α6β2γ2. Furthermore, we provide evidence for the facts that in the Xenopus oocyte (a) the formation of the different receptor types depends on the relative abundance of cRNAs coding for the different receptor subunits and (b) that functional dual subunit combinations αβ do not form in the presence of cRNA coding for the γ subunit.
Disruption of the Epilepsy KCNQ2 Gene Results in Neural Hyperexcitability
Authors: Watanabe Hirotaka , Nagata Eiichiro , Kosakai Arifumi , Nakamura Motonao , Yokoyama Masahiro , Tanaka Kortaro , Sasai Hitoshi
Published: March 2003

Abstract : Benign familial neonatal convulsion (BFNC) is a common idiopathic epilepsy with autosomal dominant inheritance. Recently, two novel voltage‐dependent potassium channel genes, KCNQ2 and KCNQ3, were identified by positional cloning as being responsible for BFNC. Heterotetramers of the products of these genes form M‐channels and regulate the threshold of electrical excitability of neurons. We disrupted the mouse KCNQ2 gene via gene targeting to study the relationship between KCNQ2 and epilepsy. Homozygous pups (KCNQ2 ‐/‐) died within a few hours after birth owing to pulmonary atelectasis that was not due to the status of epileptic seizures, although their development was morphologically normal. Heterozygous mice had decreased expression of KCNQ2 and showed hypersensitivity to pentylenetetrazole, an inducer of seizure. These data indicate that the decreased expression of KCNQ2 might cause a hyperexcitability of the CNS, which accounts for the mechanism of BFNC.
trans‐Resveratrol Protects Embryonic Mesencephalic Cells from tert‐Butyl Hydroperoxide
Authors: Karlsson Jenny , Emgard Mia , Brundin Patrik , Burkitt Mark J.
Published: March 2003

Abstract : In recent years, the antioxidant and other

pharmacological properties of resveratrol, a natural product present in grapes

and wine, have attracted considerable interest from the biomedical research

community. In an examination of the potential neuroprotective properties of

the compound, we have investigated the ability of resveratrol to protect rat

embryonic mesencephalic tissue, rich in dopaminergic neurones, from the

prooxidant tert‐butyl hydroperoxide. Using the electron paramagnetic

resonance (EPR) spin‐trapping technique, the main radicals detected in cell

suspensions were the tert‐butoxyl radical and the methyl radical,

indicating the one‐electron reduction of the peroxide followed by a

β‐scission reaction. The appearance of EPR signals from the trapped

radicals preceded the onset of cytotoxicity, which was almost exclusively

necrotic in nature. The inclusion of resveratrol in incubations resulted in

the marked protection of cells from tert‐butyl hydroperoxide. In parallel spin‐trapping experiments, we were able to demonstrate the scavenging of radicals by resveratrol, which involved direct competition between resveratrol and the spin trap for reaction with the radicals. To our knowledge, this is the first example in which cytoprotection by resveratrol has been demonstrated by EPR spin‐trapping competition kinetics to be due to its scavenging of the radicals responsible for the toxicity of a prooxidant.
Dopamine D1 Receptor‐Induced Gene Transcription Is Modulated by DARPP‐32
Authors: Svenningsson Per , Fienberg Allen A. , Allen Patrick B. , Moine Catherine Le , Lindskog Maria , Fisone Gilberto , Greengard Paul , Fredholm Bertil B.
Published: March 2003

Abstract : The role of the dopamine‐ and cyclic AMP‐regulated

phosphoprotein of Mr 32,000 (DARPP‐32) in dopaminergic regulation

of gene transcription in striatum and globus pallidus was examined. Mice with

targeted disruption of the gene encoding DARPP‐32, its homologue, inhibitor‐1,

or both, were used. Pharmacological characterization showed that mutant mice

had normal basal levels of dopamine D1 and D2 receptors

and adenosine A2A receptors. Basal expression levels of the

striatonigral‐specific neuropeptides substance P and prodynorphin and the

immediate early genes c‐fos and NGFI‐A were also unaltered in mutant

mice. A full D1 receptor agonist, SKF 82958, up‐regulated the

expression of these neuropeptides and immediate early genes significantly more

in wild‐type mice than in mice lacking DARPP‐32. Moreover, the additive

stimulation of SKF 82958 and quinelorane, a D2 receptor agonist, on

c‐fos mRNA in globus pallidus was significantly decreased in DARPP‐32 and DARPP‐32/I‐1 knockout mice. No changes in dopamine receptor‐induced gene expression were found in I‐1 knockout mice. These results demonstrate an important involvement of DARPP‐32 in dopamine receptor‐mediated regulation of gene expression both in striatal neurons, which are enriched in DARPP‐32, and in pallidal neurons, which do not contain DARPP‐32.
Mechanisms of 5‐Aminolevulinic Acid Uptake at the Choroid Plexus
Authors: Novotny A. , Xiang J. , Stummer W. , Teuscher N. S. , Smith D. E. , Keep R. F.
Published: March 2003

Abstract : 5‐Aminolevulinic acid (5‐ALA) is a precursor of

porphyrins and heme that has been implicated in the neuropsychiatric symptoms

associated with porphyrias. It is also being used clinically to delineate

malignant gliomas. The blood‐CSF barrier may be an important interface for

5‐ALA transport between blood and brain as in vivo studies have indicated

5‐ALA is taken up by the choroid plexuses whereas the normal blood‐brain

barrier appears to be relatively impermeable. This study examines the

mechanisms of 5‐[3H]ALA uptake into isolated rat lateral ventricle

choroid plexuses. Results suggest that there are two uptake mechanisms. The

first was a Na+‐independent uptake system that was pH dependent

(being stimulated at low pH). Uptake was inhibited by the dipeptide Gly‐Gly

and by cefadroxil, an α‐amino‐containing cephalosporin. These properties

are the same as the proton‐dependent peptide transporters PEPT1 and PEPT2,

which have recently been shown to transport 5‐ALA in frog oocyte expression

experiments. Choroid plexus uptake was not inhibited by captopril, a PEPT1

inhibitor, suggesting PEPT2‐mediated uptake. The presence of PEPT2 and absence

of PEPT1 in the choroid plexus were confirmed by western blotting. The second

potential mechanism was both Na+ and HCO3‐

dependent and appears to be an organic anion transporter, although it is

possible that removal of Na+ and HCO3‐ may

indirectly affect PEPT2 by affecting intracellular pH. The presence of PEPT2

and a putative Na+/HCO3‐‐dependent organic anion transporter is important not only for an understanding of 5‐ALA movement between blood and brain but also because these transporters may affect the distribution of a number of drugs between blood and CSF.
Dexamethasone Induces Lipocalin‐Type Prostaglandin D Synthase Gene Expression in Mouse Neuronal Cells
Authors: García‐Fernández Luis F , Iñiguez Miguel Angel , Eguchi Naomi , Fresno Manuel , Urade Yoshihiro , Muñoz Alberto
Published: March 2003

Lipocalin‐type prostaglandin (PG) D synthase (L‐PGDS) is responsible for the production of PGD2, the main PG in the CNS. PGD2 is

an endogenous sleep inducer, and it is involved in the control of odor and

pain responses and body temperature. In addition, PGD synthase transports

lipophilic molecules in the subarachnoid space and CSF. By northern and

western assays we show that the synthetic glucocorticoid dexamethasone, an

inhibitor of PG production in most tissues, induces L‐PGDS mRNA and protein in

a dose‐ and time‐dependent fashion in mouse neuronal GT1‐7 cells. Accordingly,

dexamethasone increases cellular L‐PGDS enzymatic activity. Dexamethasone

induced L‐PGDS gene transcription in run‐on assays and activated the mouse

L‐PGDS gene promoter in transiently transfected cells. It is interesting that

the tumor promoter 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA), which induces the synthesis of PGs in many tissues, inhibited the increase in L‐PGDS expression induced by dexamethasone. In contrast, neither dexamethasone nor TPA affected the expression of cyclooxygenases‐1 and ‐2. Our data demonstrate that dexamethasone induces L‐PGDS gene transcription in neuronal cells.
A Pathway of Neuronal Apoptosis Induced by Hypoxia/Reoxygenation
Authors: Tamatani Michio , Mitsuda Noriaki , Matsuzaki Hideo , Okado Haruo , Miyake Shin‐ichi , Vitek Michael P , Yamaguchi Atsushi , Tohyama Masaya
Published: March 2003

As a model of the reperfusion injury found in stroke, we have exposed neurons to hypoxia followed by reoxygenation. Neurons treated with hypoxia/reoxygenation (H/R) respond by activating nuclear factor‐κB (NFκB), releasing cytochrome c from their mitochondria, and

ultimately dying. Further supporting an apoptotic mechanism, expression of the

antiapoptotic Bcl‐2 and Bcl‐x proteins was increased following H/R. In this

model, adenoviral‐mediated transduction of lκB expression inhibited

NFκB activation and significantly accelerated cytochrome c

release and caspase‐dependent neuronal death. At the same time, expression of

mutated lκB prevented the increased expression of endogenous Bcl‐2 and

Bcl‐x. In the presence of mutated lκB, singular overexpression of only

Bcl‐2 by adenoviral‐mediated transduction significantly inhibited cytochrome

c release, caspase‐3‐like activation, and cell death in response to H/R. These findings suggest a pathway where NFκB activation induces overexpression of Bcl‐2 and Bcl‐x, which function to prevent apoptotic cell death following H/R treatments.
Correlation Between Caspase‐3 Activation and Three Different Markers of DNA Damage in Neonatal Cerebral Hypoxia‐Ischemia
Authors: Zhu Changlian , Wang Xiaoyang , Hagberg Henrik , Blomgren Klas
Published: March 2003

Caspase‐3 has been identified as a key protease that, by targeting a limited number of proteins, can disrupt essential homeostatic processes and initiate an orderly disassembly of cells, including degradation of genomic DNA. We demonstrate the usefulness of an antibody specific for activated caspase‐3 in a model of neonatal rat hypoxia‐ischemia (Hl) and correlate the spatial and temporal activation of caspase‐3 with three different markers of DNA damage and with the loss of a neuronal marker [microtubule‐associated protein 2 (MAP 2)]. An oligonucleotide hairpin probe (HPP) with one base overhang in the 3′ end displayed a close colocalization with caspase‐3 activation at 3 h post‐Hl, whereas terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling (TUNEL) appeared later (24 h post‐Hl). A monoclonal antibody against single‐stranded DNA appeared to stain an entirely different population of cells, not positive for active caspase‐3, HPP, or TUNEL at this time point. After 24 h of reperfusion, however, when cellular injury is extensive, all markers stained a large number of cells with a high degree of colocalization, and all markers delineated regions with loss of MAP 2. We conclude that the HPP shows the best correlation with pathological caspase‐3 activation in this model.
Real‐Time Analysis of Preprotachykinin Promoter Activity in Single Cortical Neurons
Authors: Walker Paul D , Andrade Rodrigo , Quinn John P , Bannon Michael J
Published: March 2003

Technological limitations have hindered the study of gene elements regulating transcription within CNS neurons. In the present stuides, rat cortical brain slices endogenously expressing the preprotachykinin (PPT) gene were transfected with gene constructs encompassing green fluorescent protein (GFP) under the control of the PPT promoter. These slices were maintained in organotypic culture so that the fluorescence intensity within individual living cells could be quantified using laser scanning confocal microscopy before and after application of stimulatory agents. Combined treatment with forskolin and elevated potassium significantly increased expression of both endogenous PPT mRNA and the PPT promoter‐GFP construct. The ability to follow fluorescence changes within single neurons in real time offers a powerful “within‐subject” experimental approach for analysis of neural gene promoters.
Zebrafish Genes for Neuropeptide Y and Peptide YY Reveal Origin by Chromosome Duplication from an Ancestral Gene Linked to the Homeobox Cluster
Authors: Söderberg Charlotte , Wraith Amanda , Ringvall Maria , Yan Yi‐Lin , Postlethwait John H. , Brodin Lennart , Larhammar Dan
Published: March 2003

Abstract: Neuropeptide Y (NPY) and peptide YY (PYY) are related

36‐amino acid peptides. NPY is widely distributed in the nervous system and

has several physiological roles. PYY serves as an intestinal hormone as well

as a neuropeptide. We report here cloning of the npy and pyy

genes in zebrafish (Danio rerio). NPY differs at only one to four

amino acid positions from NPY in other jawed vertebrates. Zebrafish PYY

differs at three positions from PYY from other fishes and at 10 positions from

mammals. In situ hybridization showed that neurons containing NPY mRNA have a

widespread distribution in the brain, particularly in the telencephalon, optic

tectum, and rhombencephalon. PYY mRNA was found mainly in brainstem neurons,

as reported previously for vertebrates as divergent as the rat and the

lamprey, suggesting an essential role for PYY in these neurons. PYY mRNA was

observed also in the telencephalon. These results were confirmed by

immunocytochemistry. As in the human, the npy gene is located

adjacent to homeobox (hox) gene cluster A (copy a in zebrafish),

whereas the pyy gene is located close to hoxBa. This

suggests that npy and pyy arose from a common ancestral gene

in a chromosomal duplication event that also involved the hox gene

clusters. As zebrafish has seven hox clusters, it is possible that additional NPY family genes exist or have existed. Also, the NPY receptor system seems to be more complex in zebrafish than in mammals, with at least two receptor genes without known mammalian orthologues.
Sequential Treatment of SH‐SY5Y Cells with Retinoic Acid and Brain‐Derived Neurotrophic Factor Gives Rise to Fully Differentiated, Neurotrophic Factor‐Dependent, Human Neuron‐Like Cells
Authors: Encinas Mario , Iglesias Montse , Liu Yuhui , Wang Hongyin , Muhaisen Ashraf , Ceña Valentin , Gallego Carme , Comella Joan X.
Published: March 2003

Abstract: A rapid and simple procedure is presented to obtain nearly pure populations of human neuron‐like cells from the SH‐SY5Y neuroblastoma cell line. Sequential exposure of SH‐SY5Y cells to retinoic acid and brain‐derived neurotrophic factor in serum‐free medium yields homogeneous populations of cells with neuronal morphology, avoiding the presence of other neural crest derivatives that would normally arise from those cells. Cells are withdrawn from the cell cycle, as shown by 5‐bromo‐2′‐deoxyuridine uptake and retinoblastoma hypophosphorylation. Cell survival is dependent on the continuous presence of brain‐derived neurotrophic factor, and removal of this neurotrophin causes apoptotic cell death accompanied by an attempt to reenter the cell cycle. Differentiated cells express neuronal markers, including neurofilaments, neuron‐specific enolase, and growth‐associated protein‐43 as well as neuronal polarity markers such as tau and microtubule‐associated protein 2. Moreover, differentiated cultures do not contain glial cells, as could be evidenced after the negative staining for glial fibrillary acidic protein. In conclusion, the protocol presented herein yields homogeneous populations of human neuronal differentiated cells that present many of the characteristics of primary cultures of neurons. This model may be useful to perform large‐scale biochemical and molecular studies due to its susceptibility to genetic manipulation and the availability of an unlimited amount of cells.
The GLT‐1 and GLAST Glutamate Transporters Are Expressed on Morphologically Distinct Astrocytes and Regulated by Neuronal Activity in Primary Hippocampal Cocultures
Authors: Perego C. , Vanoni C. , Bossi M. , Massari S. , Basudev H. , Longhi R. , Pietrini G.
Published: March 2003

Abstract: The GLT‐1 and GLAST astroglial transporters are the glutamate transporters mainly involved in maintaining physiological extracellular glutamate concentrations. Defects in neurotransmitter glutamate transport may represent an important component of glutamate‐induced neurodegenerative disorders (such as amyotrophic lateral sclerosis) and CNS insults (ischemia and epilepsy). We characterized the protein expression of GLT‐1 and GLAST in primary astrocyte—neuron cocultures derived from rat hippocampal tissues during neuron differentiation/maturation. GLT‐1 and GLAST are expressed by morphologically distinct glial fibrillary acidic protein‐positive astrocytes, and their expression correlates with the status of neuron differentiation/maturation and activity. Up‐regulation of the transporters paralleled the content of the synaptophysin synaptic vesicle marker p38, and down‐regulation was a consequence of glutamate‐induced neuronal death or the reduction of synaptic activity. Finally, soluble factors in neuronal‐conditioned media prevented the down‐regulation of the GLT‐1 and GLAST proteins. Although other mechanisms may participate in regulating GLT‐1 and GLAST in the CNS, our data indicate that soluble factors dependent on neuronal activity play a major regulating role in hippocampal cocultures.
MPTP Activates c‐Jun NH2‐Terminal Kinase (JNK) and Its Upstream Regulatory Kinase MKK4 in Nigrostriatal Neurons In Vivo
Authors: Saporito Michael S. , Thomas Beth Ann , Scott Richard W.
Published: March 2003

Abstract: The neuropathology of Parkinson's disease is reflected in

experimental animals treated with the selective nigrostriatal dopaminergic

neurotoxin MPTP. Neurons exposed to MPTP (MPP+) express

morphological features of apoptosis, although the intracellular pathways that

produce this morphology have not been established. The c‐Jun

NH2‐terminal kinase (JNK) signaling cascade has been implicated as

a mediator of MPTP‐induced apoptotic neuronal death based on the ability of

CEP‐1347/KT‐7515, an inhibitor of JNK activation, to attenuate MPTP‐induced

nigrostriatal dopaminergic degeneration. In these studies, MPTP‐mediated

activation of the JNK signaling pathway was assessed in the nigrostriatal

system of MPTP‐treated mice. MPTP elevated levels of phosphorylated JNK and

JNK kinase (MKK4; also known as SEK1 or JNKK), by 2.5‐ and fivefold,

respectively. Peak elevations occurred soon after administration of MPTP and

coincided with peak CNS levels of MPP+. Increased MKK4

phosphorylation, but not JNK phosphorylation, was found in the striatum,

suggesting that activation of MKK4 occurs in injured dopaminergic terminals.

Both JNK and MKK4 phosphorylations were attenuated by pretreatment with

l‐deprenyl, indicating that these phosphorylation events were

mediated by MPP+. Moreover, CEP‐1347/KT‐7515 inhibited MPTP‐mediated MKK4 and JNK signaling at a dose that attenuates MPTP‐induced dopaminergic loss. These data implicate this signaling pathway in MPTP‐mediated nigrostriatal dopaminergic death and suggest that it may be activated in the degenerative process in Parkinson's disease.
Estrogen Enhances Uptake of Amyloid β‐Protein by Microglia Derived from the Human Cortex
Authors: Li Rena , Shen Yong , Yang Li‐Bang , Lue Lih‐Fen , Finch Caleb , Rogers Joseph
Published: March 2003

Abstract: In recent years, inflammatory mechanisms have been
increasingly appreciated as important steps in the pathology of Alzheimer's
disease (AD). There are two pathological defects in AD: chronic inflammation
and impaired clearance of amyloid β‐peptide (Aβ). In the periphery,
estrogen both increases macrophage phagocytosis and has antiinflammatory
effects. If estrogen had a similar effect in the CNS, it could reverse
inflammatory defects in AD. Although microglia are a key component of the
immune system and help clear Aβ deposits in the AD brain, little is known
about the effects of estrogen on CNS microglia. Therefore, we sought to
determine the relationship between estrogen treatment and internalization of
Aβ by microglia by quantifying the internalization of aggregated Aβ
by human cortical microglia. Aβ uptake was found to be dose‐ and
time‐dependent in cultured microglia. Increased Aβ uptake was observed at
1.5 and 24 h after addition of aggregated Aβ (50, 100, or 1,000
nM Aβ), and this uptake was enhanced by pretreatment with estrogen. The expression of estrogen receptor (ER) β (ER‐β) was also up‐regulated by estrogen treatment. Cells cotreated with ICI 182,780, an ER antagonist, showed significantly reduced internalization of Aβ in cultured microglia. These results indicate that microglia express an ER‐β but that the effect of estrogen on enhancing clearance of Aβ may be related to the receptor‐independent action of estrogen or to nonclassical ER effects of estrogen. Thus, stimulation of the ER might contribute to the therapeutic action of estrogen in the treatment of AD.
Defective Prodynorphin Processing in Mice Lacking Prohormone Convertase PC2
Authors: Berman Y. , Mzhavia N. , Polonskaia A. , Furuta M. , Steiner D. F. , Pintar J. E. , Devi L. A.
Published: March 2003

Abstract: Prodynorphin, a multifunctional precursor of several important opioid peptides, is expressed widely in the CNS. It is processed at specific single and paired basic sites to generate various biologically active products. Among the prohormone convertases (PCs), PC1 and PC2 are expressed widely in neuroendocrine tissues and have been proposed to be the major convertases involved in the biosynthesis of hormonal and neural peptides. In this study we have examined the physiological involvement of PC2 in the generation of dynorphin (Dyn) peptides in mice lacking active PC2 as a result of gene disruption. Enzymological and immunological assays were used to confirm the absence of active PC2 in these mice. The processing profiles of Dyn peptides extracted from brains of these mice reveal a complete lack of Dyn A‐8 and a substantial reduction in the levels of Dyn A‐17 and Dyn B‐13. Thus, PC2 appears to be involved in monobasic processing, leading to the generation of Dyn A‐8, Dyn A‐17, and Dyn B‐13 from prodynorphin under physiological conditions. Brains of heterozygous mice exhibit only half the PC2 activity of wild‐type mice; however, the levels of Dyn peptides in these mice are similar to those of wild‐type mice, suggesting that a 50% reduction in PC2 activity is not sufficient to significantly reduce prodynorphin processing. The disruption of the PC2 gene does not lead to compensatory up‐regulation in the levels of other convertases with similar substrate specificity because we find no significant changes in the levels of PC1, PC5/PC6, or furin in these mice as compared with wild‐type mice. Taken together, these results support a critical role for PC2 in the generation of Dyn peptides.
Novel CNS Glutamate Receptor Subunit Genes of Drosophila melanogaster
Authors: Völkner Michaela , Lenz‐Böhme Bettina , Betz Heinrich , Schmitt Bertram
Published: March 2003

Abstract: We report the identification and characterization of two
genes from Drosophila melanogaster that encode novel ionotropic
glutamate receptor proteins, named DGluR‐IB and DNMDAR‐II, and that are
located on chromosome 3L, region 67AB, and the X chromosome, position 2B,
respectively. The DGluR‐IB full‐length cDNA was isolated from
Drosophila embryonic and head libraries. The encoded protein of 1,095 amino acids displays high sequence identity (73%) to DGluR‐IA. The DNMDAR‐II gene was identified by sequence‐homology searches in databases. The deduced protein shows moderate sequence identity (29‐31%) to the mouse NMDAR2A‐D receptor subunits. Whole‐mount in situ hybridization on embryos revealed DGluR‐IB and DNMDAR‐II transcripts in the CNS. Immunofluorescence analysis of the adult fly brain indicates that the DGluR‐IB protein is expressed in neurons implicated in the regulation of the circadian clock.
Thyroid Hormone‐Induced Morphological Differentiation and Maturation of Astrocytes Are Mediated Through the β‐Adrenergic Receptor
Authors: Gharami Kusumika , Das Sumantra
Published: March 2003

Abstract: The molecular mechanisms associated with thyroid hormone
(TH)‐induced maturation of astrocytes have been studied using primary
cultures. We have previously demonstrated that unlike normal astrocyte
cultures, hypothyroid cultures fail to differentiate from flat polygonal cells
with epithelioid morphology into mature process‐bearing cells with stellate
morphology. Addition of TH to the hypothyroid cells reverses the effect, and
astrocytes transform into stellate cells. The β‐adrenergic receptor
(β‐AR) agonist isoproterenol (ISP) has a similar effect, whereas
simultaneous addition of the β‐adrenergic antagonist propranolol blocks
the differentiation induced by TH or ISP. Addition of TH or ISP to hypothyroid
cultures is also associated with a decrease in the level of filamentous
cytoskeletal (Fi) actin and an increase in the level of actin mRNA.
Although addition of propranolol inhibited the decline in the level of
Fi actin in the TH‐ or ISP‐supplemented cells as well as the
induction of actin mRNA by TH, it partially inhibited the ISP‐induced actin
mRNA in these cultures. The hormone‐induced maturation appears to be
selectively regulated through the β2‐AR. The overall results indicate that the β‐adrenergic system plays an obligatory role in promoting TH‐induced differentiation and maturation of astrocytes and in regulating the hormone‐induced expression of actin and its intracellular organization in a way conducive to the morphological differentiation of the cells.
Brain‐Derived Neurotrophic Factor, Neurotrophin‐3, and Neurotrophin‐4/5 Prevent the Death of Striatal Projection Neurons in a Rodent Model of Huntington's Disease
Authors: Pérez‐Navarro Esther , Canudas Anna M. , Åkerud Peter , Alberch Jordi , Arenas Ernest
Published: March 2003

Abstract: Intrastriatal injection of quinolinate has been proven to be a very useful animal model to study the pathogenesis and treatment of Huntington's disease. To determine whether growth factors of the neurotrophin family are able to prevent the degeneration of striatal projection neurons, cell lines expressing brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), or neurotrophin‐4/5 (NT‐4/5) were grafted in the adult rat striatum before quinolinate injection. Three days after lesioning, ongoing cell death was assessed by in situ detection of DNA fragmentation. In animals grafted with the control cell line, quinolinate injection induced a gradual cell loss that was differentially prevented by intrastriatal grafting of BDNF‐, NT‐3‐, or NT‐4/5‐secreting cells. Seven days after lesioning, we characterized striatal projection neurons that were protected by neurotrophins. Quinolinate injection, alone or in combination with the control cell line, induced a selective loss of striatal projection neurons. Grafting of a BDNF‐secreting cell line prevented the loss of all types of striatal projection neurons analyzed. Glutamic acid decarboxylase 67‐, preproenkephalin‐, and preprotachykinin A‐ but not prodynorphin‐expressing neurons were protected by grafting of NT‐3‐ or NT‐4/5‐secreting cells but with less efficiency than the BDNF‐secreting cells. Our findings show that neurotrophins are able to promote the survival of striatal projection neurons in vivo and suggest that BDNF might be beneficial for the treatment of striatonigral degenerative disorders, including Huntington's disease.