bluuebunny/crossref_metadata_2025 huggingface dataset:
The FTDP‐17‐Linked Mutation R406W Abolishes the Interaction of Phosphorylated Tau with Microtubules
Authors: Pérez M. , Lim F. , Arrasate M. , Avila J.
Published: March 2003

Abstract: The recent finding that several point mutations in the gene encoding for the microtubule‐binding protein tau correlate with neurological disorders has heightened interest in the mechanisms of destabilization of this protein. In this study the functional consequences of the tau mutation R406W on the interaction of the protein with microtubules have been analyzed. Mutated tau is less phosphorylated than its normal counterpart at serines 396 and 404. Furthermore, the phosphorylated mutant protein is unable to bind to microtubules, and, as a consequence, microtubules assembled after transient nocodazole treatment in the presence of this tau variant contain only unmodified tau and appear to form more and longer bundles than those assembled in the presence of wild‐type tau. We propose that phosphorylated tau, unbound to microtubules, could accumulate in the cytoplasm.
Neuronal Apoptosis Induced by Pharmacological Concentrations of 3‐Hydroxykynurenine
Authors: Wei Huafeng , Leeds Peter , Chen Ren‐Wu , Wei Wenlin , Leng Yan , Bredesen Dale E. , Chuang De‐Maw
Published: March 2003

Abstract : We have studied neurotoxicity induced by pharmacological

concentrations of 3‐hydroxykynurenine (3‐HK), an endogenous toxin implicated

in certain neurodegenerative diseases, in cerebellar granule cells, PC12

pheochromocytoma cells, and GT1‐7 hypothalamic neurosecretory cells. In all

three cell types, the toxicity was induced in a dose‐dependent manner by 3‐HK

at high micromolar concentrations and had features characteristic of

apoptosis, including chromatin condensation and internucleosomal DNA cleavage.

In cerebellar granule cells, the 3‐HK neurotoxicity was unaffected by xanthine

oxidase inhibitors but markedly potentiated by superoxide dismutase and its

hemelike mimetic, MnTBAP [manganese(III) tetrakis(benzoic acid)porphyrin

chloride]. Catalase blocked 3‐HK neurotoxicity in the absence and presence of

superoxide dismutase or MnTBAP. The formation of H2O2

was demonstrated in PC12 and GT1‐7 cells treated with 3‐HK, by measuring the

increase in the fluorescent product, 2′,7′‐dichlorofluorescein. In

both PC12 and cerebellar granule cells, inhibitors of the neutral amino acid

transporter that mediates the uptake of 3‐HK failed to block 3‐HK toxicity.

However, their toxicity was slightly potentiated by the iron chelator,

deferoxamine. Taken together, our results suggest that neurotoxicity induced

by pharmacological concentrations of 3‐HK in these cell types is mediated

primarily by H2O2, which is formed most likely by auto‐oxidation of 3‐HK in extracellular compartments. 3‐HK‐induced death of PC12 and GT1‐7 cells was protected by dantrolene, an inhibitor of calcium release from the endoplasmic reticulum. The protection by dantrolene was associated with a marked increase in the protein level of Bcl‐2, a prominent antiapoptotic gene product. Moreover, overexpression of Bcl‐2 in GT1‐7 cells elicited by gene transfection suppressed 3‐HK toxicity. Thus, dantrolene may elicit its neuroprotective effects by mechanisms involving up‐regulation of the level and function of Bcl‐2 protein.
Effects of 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine on Differentiating Mouse N2a Neuroblastoma Cells
Authors: De Girolamo Luigi A. , Billett E. Ellen , Hargreaves Alan J.
Published: March 2003

Abstract : The effect of the neurotoxin

1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) was investigated in mouse

N2a neuroblastoma cells, induced to differentiate by serum withdrawal and

addition of dibutyryl cyclic AMP, over a 24‐h period. Addition of MPTP (10

μM) during differentiation caused a change in cell morphology characterised by an inhibition of axon outgrowth, in the absence of cell death. Biochemical characterisation by western blotting revealed that MPTP had no significant effects on the levels of actin, α‐tubulin, or total heavy‐chain neurofilament (NF‐H). However, NF‐H phosphorylation appeared to increase following MPTP treatment when blots were probed with the phosphorylation state‐specific antibodies RMd09 and Ta51. In addition, indirect immunofluorescence analysis revealed an accumulation of phosphorylated NF‐H in the cell perikaryon, suggesting that altered NF‐H distribution was associated with the observed effects of MPTP on cell morphology. These changes may represent a useful in vitro marker of MPTP neurotoxicity within a simple differentiating neuronal cell model system.
Neuroblastoma Cells Expressing the Noradrenaline Transporter Are Destroyed More Selectively by 6‐Fluorodopamine than by 6‐Hydroxydopamine
Authors: Seitz Gabriele , Stegmann Hartmut B , Jäger Heidrun H , Schlude Hans M , Wolburg Hartwig , Roginsky Vitaly A , Niethammer Dietrich , Bruchelt Gernot
Published: March 2003

6‐Hydroxydopamine (6‐OHDA) has been used for lesioning catecholaminergic neurons and attempted purging of neuroblastoma cells from hematopoietic stem cells in autologous bone marrow transplantation (ABMT). Neurotoxicity is mediated primarily by reactive oxygen species. In ABMT, 6‐OHDA, as a purging agent, has been unsuccessful. At physiological pH it autooxidizes before targeted uptake, resulting in nonspecific cytotoxicity of nontarget cells. A catecholamine analogue, similar to 6‐OHDA but with a lower rate of autooxidation enabling uptake by target cells, is thus required. Electron paramagnetic resonance spectra in this study show that 6‐fluorodopamine (6‐FDA) hydrolyzes slowly to 6‐OHDA at physiological pH. Oxygen consumption, H2O2, and quinone production are found to be

intermediate between those of 6‐OHDA and dopamine (DA). Relative neurotoxicity

of these compounds was assessed by cell viability and DNA damage in the human

neuroblastoma lines SH‐SY5Y and SK‐N‐LO, which express and lack the

noradrenaline transporter, respectively. Specific uptake of DA and 6‐FDA by

SH‐SY5Y cells was demonstrated by competitive

m‐[131I]iodobenzylguanidine uptake inhibition. The competition by 6‐OHDA was low owing to rapid autooxidation during incubation with equal toxicity toward both cell types. 6‐FDA toxicity was preferential for SH‐SY5Y cells and reduced in the presence of desipramine, a catecholamine uptake inhibitor. We demonstrate that 6‐FDA cytotoxicity is more specific for cells expressing catecholamine reuptake systems than is 6‐OHDA cytotoxicity.
Identification of a Histamine H3‐like Receptor in the Zebrafish (Danio rerio) Brain
Authors: Peitsaro Nina , Anichtchik Oleg V , Panula Pertti
Published: March 2003

The distribution of histaminergic fibers in the zebrafish brain was recently shown to resemble that in mammals. Expression of l‐histidine decarboxylase (HDC) mRNA was shown only in the area

corresponding to that expressing HDC in mammals. This indicates that the

zebrafish could be a useful model for studies on the function of the brain

histaminergic system. In this study an H3‐like receptor is

identified in zebrafish brain. With binding studies using

N‐α‐[3H]methylhistamine on zebrafish brain sections,

signals were observed in several regions. Highest densities were detected in

optic tectum and hypothalamus. The autoradiographic signal was abolished

completely by the H3‐specific antagonist clobenpropit and

significantly reduced by another H3 antagonist, thioperamide.

Histamine and immepip induced an increase of guanosine

5′‐(γ‐[35S]thio)triphosphate binding in several areas

of the zebrafish brain. The activation was blocked with clobenpropit but not

with cimetidine or mepyramine. These results indicate that the zebrafish has a

histamine H3‐like receptor that functionally interacts with the

inhibitory, Gi/Go, class of G proteins. No previous

evidence for a histamine receptor in zebrafish exists. The receptor described

here is apparently similar to the mammalian H3 receptor, making

this the first description of a histamine H3‐like receptor in a lower vertebrate.
Preconditioning with Cortical Spreading Depression Decreases Intraischemic Cerebral Glutamate Levels and Down‐Regulates Excitatory Amino Acid Transporters EAAT1 and EAAT2 from Rat Cerebal Cortex Plasma Membranes
Authors: Douen Andre G , Akiyama Katsunori , Hogan Matthew J , Wang Fuhu , Dong Li , Chow Ava K , Hakim Antoine
Published: March 2003

We previously reported a 50% reduction in cortical infarct volume following transient focal cerebral ischemia in rats preconditioned 3 days earlier with cortical spreading depression (CSD). The mechanism of the protective effect of prior CSD remains unknown. Recent studies demonstrate reversal of excitatory amino acid transporters (EAATs) to be a principal cause for elevated extracellular glutamate levels during cerebral ischemia. The present study measured the effect of CSD preconditioning on (a) intraischemic glutamate levels and (b) regulation of glutamate transporters within the ischemic cortex of the rat. Three days following either CSD or sham preconditioning, rats were subjected to 200 min of focal cerebral ischemia, and extracellular glutamate concentration was measured by in vivo microdialysis. Cortical glutamate exposure decreased 70% from 1,772.4 ± 1,469.2 μM‐min in

sham‐treated (n = 8) to 569.0 ± 707.8 μM‐min in CSD‐treated

(n = 13) rats (p <0.05). The effect of CSD preconditioning on glutamate transporter levels in plasma membranes (PMs) prepared from rat cerebral cortex was assessed by western blot analysis. Down‐regulation of the glial glutamate transporter isoforms EAAT2 and EAAT1 from the PM fraction was observed at 1, 3, and 7 days but not at 0 or 21 days after CSD. Semiquantitative lane analysis showed a maximal decrease of 90% for EAAT2 and 50% for EAAT1 at 3 days post‐CSD. The neuronal isoform EAAT3 was unaffected by CSD. This period of down‐regulation coincides with the time frame reported for induced ischemic tolerance. These data are consistent with reversal of glutamate transporter function contributing to glutamate release during ischemia and suggest that down‐regulation of these transporters may contribute to ischemic tolerance induced by CSD.
The Human Brain Has Distinct Regional Expression Patterns of Estrogen Receptor α mRNA Isoforms Derived from Alternative Promoters
Authors: Österlund Marie K. , Grandien Kaj , Keller Eva , Hurd Yasmin L.
Published: March 2003

Abstract: The human estrogen receptor (ER) α gene is transcribed from multiple promoters, generating mRNA isoforms with unique 5′ ends in the untranslated region. In the present study, alternative promoters were shown to regulate the ERα gene expression in different neuronal populations of the human brain. By using in situ hybridization histochemistry, the A and B promoters, but not the C promoter, in the ERα gene were found to be active in the human forebrain. The mRNA isoform transcribed from the A promoter was expressed in low levels in most of the brain areas where ERα mRNA was present. In contrast, the B promoter mRNA isoform was more restricted, localized predominantly in high‐expressing ERα mRNA regions. The gross anatomical distribution of the different mRNA isoforms analyzed with RT‐PCR generally supported the results obtained by the in situ hybridization. Estrogen is known to modulate many different brain functions, such as neuroendocrine events associated with reproduction, mood, and cognition, likely to be mediated by different neuronal populations. Thus, the current findings of alternative ERα promoter expression in distinct neuronal populations suggest that multiple promoter usage is a possible mechanism to achieve differentiated regulation of the ERα expression, dependent on the cell phenotype and consequently the functions mediated by the specific neuron.
Overexpression of Glutathione Peroxidase Increases the Resistance of Neuronal Cells to Aβ‐Mediated Neurotoxicity
Authors: Barkats Martine , Millecamps Stéphanie , Abrioux Pascale , Geoffroy Marie‐Claude , Mallet Jacques
Published: March 2003

Abstract: Senile plaques are neuropathological manifestations in
Alzheimer's disease (AD) and are composed mainly of extracellular deposits of
amyloid β‐peptide (Aβ). Various data suggest that the accumulation
of Aβ may contribute to neuronal degeneration and that Aβ
neurotoxicity could be mediated by oxygen free radicals. Removal of free
radicals by antioxidant scavengers or enzymes was found to protect neuronal
cells in culture from Aβ toxicity. However, the nature of the free
radicals involved is still unclear. In this study, we investigated whether the
neuronal overexpression of glutathione peroxidase (GPx), the major hydrogen
peroxide (H2O2)‐de‐grading enzyme in neurons, could
increase their survival in a cellular model of Aβ‐induced neurotoxicity.
We infected pheochromocytoma (PC12) cells and rat embryonic cultured cortical
neurons with an adenoviral vector encoding GPx (Ad‐GPx) prior to exposure to
toxic concentrations of Aβ(25‐35) or (1‐40). Both PC12 and cortical
Ad‐GPx‐infected cells were significantly more resistant to Aβ‐induced
injury. These data strengthen the hypothesis of a role of
H2O2 in the mechanism of Aβ toxicity and highlight the potential of Ad‐GPx to reduce Aβ‐induced damage to neurons. These findings may have applications in gene therapy for AD.
Agonist‐Induced Internalization and Recycling of the Human A3Adenosine Receptors
Authors: Trincavelli Maria Letizia , Tuscano Daniela , Cecchetti Paolo , Falleni Alessandra , Benzi Luca , Klotz Karl‐Norbert , Gremigni Vittorio , Cattabeni Flaminio , Lucacchini Antonio , Martini Claudia
Published: March 2003

Abstract:A3adenosine receptors have been proposed to play an important role in the pathophysiology of cerebral ischemia with a regimen‐dependent nature of the therapeutic effects probably related to receptor desensitization and down‐regulation. Here we studied the agonist‐induced internalization of human A3adenosine receptors in transfected Chinese hamster ovary cells, and then we evaluated the relationship between internalization and signal desensitization and resensitization. Binding ofN6‐(4‐amino‐3‐[125I]iodobenzyl)adenosine‐5′‐N‐methyluronamide to membranes from Chinese hamster ovary cells stably transfected with the human A3adenosine receptor showed a profile typical of these receptors in other cell lines (KD= 1.3 ± 0.08 nM;Bmax= 400 ± 28 fmol/mg of proteins). The iodinated agonist, bound at 4°C to whole transfected cells, was internalized by increasing the temperature to 37°C with a rate constant of 0.04 ± 0.034 min‐1. Agonist‐induced internalization of A3adenosine receptors was directly demonstrated by immunogold electron microscopy, which revealed the localization of these receptors in plasma membranes and intracellular vesicles. Moreover, short‐term exposure of these cells to the agonist caused rapid desensitization as tested in adenylyl cyclase assays. Subsequent removal of the agonist led to restoration of the receptor function and recycling of the receptors to the cell surface. The rate constant of receptor recycling was 0.02 ± 0.0017 min‐1. Blockade of internalization and recycling demonstrated that internalization did not affect signal desensitization, whereas recycling of internalized receptors was implicated in the signal resensitization.
Purine Uptake and Release in Rat C6 Glioma Cells
Authors: Sinclair C. J. D. , LaRivière C. G. , Young James D. , Cass Carol E. , Baldwin Stephen A. , Parkinson F. E.
Published: March 2003

Abstract: Adenosine, through activation of membrane‐bound receptors,
has been reported to have neuroprotective properties during strokes or
seizures. The role of astrocytes in regulating brain interstitial adenosine
levels has not been clearly defined. We have determined the nucleoside
transporters present in rat C6 glioma cells. RT‐PCR analysis,
3H‐nucleoside uptake experiments, and
[3H]nitrobenzylthioinosine ([3H]NBMPR) binding assays
indicated that the primary functional nucleoside transporter in C6 cells was
rENT2, an equilibrative nucleoside transporter (ENT) that is relatively
insensitive to inhibition by NBMPR. [3H]Formycin B, a poorly
metabolized nucleoside analogue, was used to investigate nucleoside release
processes, and rENT2 transporters mediated [3H]formycin B release
from these cells. Adenosine release was investigated by first loading cells
with [3H]adenine to label adenine nucleotide pools. Tritium release was initiated by inhibiting glycolytic and oxidative ATP generation and thus depleting ATP levels. Our results indicate that during ATP‐depleting conditions, AMP catabolism progressed via the reactions AMP → IMP → inosine → hypoxanthine, which accounted for >90% of the evoked tritium release. It was surprising that adenosine was not released during ATP‐depleting conditions unless AMP deaminase and adenosine deaminase were inhibited. Inosine release was enhanced by inhibition of purine nucleoside phosphorylase; ENT2 transporters mediated the release of adenosine or inosine. However, inhibition of AMP deaminase/adenosine deaminase or purine nucleoside phosphorylase during ATP depletion produced release of adenosine or inosine, respectively, via the rENT2 transporter. This indicates that C6 glioma cells possess primarily rENT2 nucleoside transporters that function in adenosine uptake but that intracellular metabolism prevents the release of adenosine from these cells even during ATP‐depleting conditions.
Synaptic Activity‐Dependent Developmental Regulation of NMDA Receptor Subunit Expression in Cultured Neocortical Neurons
Authors: Hoffmann Heike , Gremme Tobias , Hatt Hanns , Gottmann Kurt
Published: March 2003

Abstract: The biophysical properties of NMDA receptors are thought
to be critical determinants involved in the regulation of long‐term synaptic
plasticity during neocortical development. NMDA receptor channel properties
are strongly dependent on the subunit composition of heteromeric NMDA
receptors. During neocortical developmentin vivo, the expression of the NMDA
receptor 2A (NR2A) subunit is up‐regulated at the mRNA and protein level
correlating with changes in the kinetic and pharmacological properties of
functional NMDA receptors. To investigate the developmental regulation of NMDA
receptor subunit expression, we studied NR2 mRNA expression in cultured
neocortical neurons. With increasing time in culture, they showed a similar
up‐regulation of NR2A mRNA expression as describedin vivo. As demonstrated by
chronic blockade of postsynaptic glutamate receptorsin vitro, the regulation
of NR2A mRNA was strongly dependent on synaptic activity. In contrast, NR2B
mRNA expression was not influenced by activity blockade. Moreover, as shown
pharmacologically, the regulation of NR2A mRNA expression was mediated by
postsynaptic Ca2+ influx through both NMDA receptors and L‐type
Ca2+ channels. It is interesting that even relatively weak expression of NR2A mRNA was correlated with clearly reduced sensitivity of NMDA receptor‐mediated whole‐cell currents against the NR2B subunit‐specific antagonist ifenprodil. Developmental changes in the expression of NR1 mRNA splice variants were also strongly dependent on synaptic activity and thus might, in addition to regulation of NR2 subunit expression, contribute to developmental changes in the properties of functional NMDA receptors. In summary, our results demonstrate that synaptic activity is a key factor in the regulation of NMDA receptor subunit expression during neocortical development.
Serotonin 5‐HT2 Receptor Stimulation of Dopamine Release in the Posterior but Not Anterior Nucleus Accumbens of the Rat
Authors: Bowers Barbara J. , Henry Miranda B. , Thielen Richard J. , McBride William J.
Published: March 2003

Abstract: The objective of the present study was to examine the
involvement of serotonin 5‐HT2 receptors within the rat nucleus
accumbens (Acc) in the regulation of dopamine (DA) release usingin vivo
microdialysis. Perfusion with the 5‐HT2 agonist
(+)‐1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane (DOI), at concentrations of
25‐250 μM, through microdialysis probes located in the posterior
Acc increased extracellular DA levels to a maximum of 200% of baseline.
DOI‐induced increases in the extracellular levels of DA were Ca2+
dependent and were inhibited by co‐perfusion with the 5‐HT2
antagonist LY‐53,857. DOI enhancement of the extracellular concentrations of
DA was observed when probes were implanted in the Acc core and shell regions
posterior to anteroposterior +1.2 mm from bregma, whereas a small reduction in
the extracellular levels of DA was observed in the anterior Acc. There were no
differences between core and shell subdivisions within either the anterior or
the posterior Acc. These results suggest that activation of 5‐HT2 receptors within the posterior, but not anterior, Acc stimulates DA release, indicating rostral‐caudal differences in the interactions of 5‐HT with DA systems in the Acc.
Effect of Transient Focal Ischemia of Mouse Brain on Energy State and NAD Levels
Authors: Paschen Wulf , Oláh Laszlo , Mies Günter
Published: March 2003

Abstract: It has been proposed that NAD depletion resulting from
excessive activation of poly(ADP‐ribose) polymerase is responsible for
secondary energy failure after transient cerebral ischemia. However, this
hypothesis has never been verified by measurement of ATP and NAD levels in the
same tissue sample. In this study, we therefore investigated the effect of
transient focal cerebral ischemia on the temporal profiles of changes in the
levels of energy metabolites and NAD. Ischemia was induced in mice by
occluding the left middle cerebral artery using the intraluminal filament
technique. Animals were subjected to 1‐h ischemia, followed by 0, 1, 3, 6, or
24 h of reperfusion. During ischemia, ATP levels, total adenylate pool, and
adenylate energy charge dropped to ∼20, 50, and 40% of control,
respectively, whereas NAD levels remained close to control. Energy state
recovered transiently, peaking at 3 h of recovery (ATP levels and total
adenylate pool recovered to 78 and 81% of control). In animals subjected to
reperfusion of varying duration, the extent of ATP depletion was clearly more
pronounced than that of NAD. The results imply that depletion of NAD pools did
not play a major role in secondary disturbances of energy‐producing metabolism
after transient focal cerebral ischemia. Changes in ATP levels were closely
related to changes in total adenylate pool (p < 0.001). The high energy charge after 6 h of reperfusion (0.90 versus a control value of 0.93) and the close relationship between the decline of ATP and total adenylate pool suggest that degradation or a washout of adenylates (owing to leaky membranes) rather than a mismatch between energy production and consumption is the main causative factor contributing to the secondary energy failure observed after prolonged recovery.
Cocaine and Methamphetamine Differentially Affect Opioid Peptide mRNA Expression in the Striatum
Authors: Adams David H. , Hanson Glen R. , Keefe Kristen A.
Published: March 2003

Abstract: In general, administration of methamphetamine and cocaine alters preprodynorphin and preproenkephalin mRNA levels in striatum. However, no study has directly compared the effects of these stimulants on opioid peptides in striatum. This study used in situ hybridization to compare directly the effects of cocaine and methamphetamine on preprodynorphin and preproenkephalin mRNAs in distinct striatal regions. Male Sprague—Dawley rats received a single administration of 15 mg/kg methamphetamine or 30 mg/kg cocaine and were killed 30 min or 3 h later. Methamphetamine and cocaine differentially affected preprodynorphin mRNA in striatum after 3 h. Densitometric analysis of film autoradiograms revealed that cocaine, but not methamphetamine, significantly increased preprodynorphin. This effect was seen throughout rostral striatum and dorsally in caudal striatum. However, specific analysis of “patches” in which preprodynorphin expression is high revealed a significantly greater effect of methamphetamine versus cocaine. In contrast, both cocaine and methamphetamine had similar effects on preproenkephalin mRNA, decreasing levels after 30 min in rostral striatum and in the core of nucleus accumbens. These data suggest that methamphetamine and cocaine have distinct postsynaptic consequences on striatal neurons.
The Transforming Growth Factor‐βs
Authors: Böttner Martina , Krieglstein Kerstin , Unsicker Klaus
Published: March 2003

Abstract: Transforming growth factor‐βs (TGF‐βs) are amongthe most widespread and versatile cytokines. Here, we first provide a briefoverview of their molecular biology, biochemistry, and signaling. We thenreview distribution and functions of the three mammalian TGF‐β isoforms,β1, β2, and β3, and their receptors in the developing and adultnervous system. Roles of TGF‐βs in the regulation of radial glia,astroglia, oligodendroglia, and microglia are addressed. Finally, we reviewthe current state of knowledge concerning the roles of TGF‐βs incontrolling neuronal performances, including the regulation of proliferationof neuronal precursors, survival/death decisions, and neuronaldifferentiation.
In Vivo Labeling of Mitochondrial Complex I (NADH:UbiquinoneOxidoreductase) in Rat Brain Using [3H]Dihydrorotenone
Authors: Talpade Deepa J. , Greene James G. , Higgins Donald S. , Greenamyre J. Timothy
Published: March 2003

Abstract: Defects in mitochondrial energy metabolism have beenimplicated in several neurodegenerative disorders. Defective complex I(NADH:ubiquinone oxidoreductase) activity plays a key role in Leber'shereditary optic neuropathy and, possibly, Parkinson's disease, but there isno way to assess this enzyme in the living brain. We previously described anin vitro quantitative autoradiographic assay using[3H]dihydrorotenone ([3H]DHR) binding to complex I. Wehave now developed an in vivo autoradiographic assay for complex I using[3H]DHR binding after intravenous administration. In vivo[3H]DHR binding was regionally heterogeneous, and brain uptake wasrapid. Binding was enriched in neurons compared with glia, and white matterhad the lowest levels of binding. In vivo [3H]DHR binding wasmarkedly reduced by local and systemic infusion of rotenone and was enhancedby local NADH administration. There was an excellent correlation betweenregional levels of in vivo [3H]DHR binding and the in vitroactivities of complex II (succinate dehydrogenase) and complex IV (cytochromeoxidase), suggesting that the stoichiometry of these components of theelectron transport chain is relatively constant across brain regions. Theability to assay complex I in vivo should provide a valuable tool toinvestigate the status of this mitochondrial enzyme in the living brain andsuggests potential imaging techniques for complex I in humans.
Hippocampal Astrocytes Exhibit Ca2+ ‐Elevating Muscarinic Cholinergic and Histaminergic Receptors In Situ
Authors: Shelton Marilee K. , McCarthy Ken D.
Published: March 2003

Abstract: Recent findings suggest that astrocytes respond to neuronally released neurotransmitters with Ca2+ elevations. These Ca2+ elevations may trigger astrocytes to release glutamate, affecting neuronal activity. Neuronal activity is also affected by modulatory neurotransmitters that stimulate G protein‐coupled receptors. These neurotransmitters, including acetylcholine and histamine, might affect neuronal activity by triggering Ca2+ ‐dependent release of neurotransmitters from astrocytes. However, there is no physiological evidence for histaminergic or cholinergic receptors on astrocytes in situ. We asked whether astrocytes have these receptors by imaging Ca2+ ‐sensitive dyes sequestered by astrocytes in hippocampal slices. Our results show that immunocytochemically identified astrocytes respond to carbachol and histamine with increases in intracellular free Ca2+ concentration. The H1 histamine receptor antagonist chlorpheniramine inhibited responses to histamine. Similarly, atropine and the M1‐selective muscarinic receptor antagonist pirenzepine inhibited carbachol‐elicited responses. Astrocyte responses to histamine and carbachol were compared with responses elicited by α1‐adrenergic and metabotropic glutamate receptor agonists. Individual astrocytes responded to different subsets of receptor agonists. Ca2+ oscillations were the prevalent response pattern only with metabotropic glutamate receptor stimulation. Finally, functional α1‐adrenergic receptors and muscarinic receptors were not detected before postnatal day 8. Our data show that astrocytes have acetylcholine and histamine receptors coupled to Ca2+. Given that Ca2+ elevations in astrocytes trigger neurotransmitter release, it is possible that these astrocyte receptors modulate neuronal activity.
Brain‐Derived Neurotrophic Factor Induces Excitotoxic Sensitivity in Cultured Embryonic Rat Spinal Motor Neurons Through Activation of the Phosphatidylinositol 3‐Kinase Pathway
Authors: Fryer Hugh J. L. , Wolf Daniel H. , Knox Ronald J. , Strittmatter Stephen M. , Pennica Diane , O'Leary Rhona M. , Russell David S. , Kalb Robert G.
Published: March 2003

Abstract:Neurotrophic factors (NTFs) can protect against or sensitize neurons to excitotoxicity. We studied the role played by various NTFs in the excitotoxic death of purified embryonic rat motor neurons. Motor neurons cultured in brain‐derived neurotrophic factor, but not neurotrophin 3, glial‐derived neurotrophic factor, or cardiotrophin 1, were sensitive to excitotoxic insult. BDNF also induces excitotoxic sensitivity (ES) in motor neurons when BDNF is combined with these other NTFs. The effect of BDNF depends on de novo protein and mRNA synthesis. Reagents that either activate or inhibit the 75‐kDa NTF receptor p75NTRdo not affect BDNF‐induced ES. The low EC50for BDNF‐induced survival and ES suggests that TrkB mediates both of these biological activities. BDNF does not alter glutamate‐evoked rises of intracellular Ca2+, suggesting BDNF acts downstream. Both wortmannin and LY294002, which specifically block the phosphatidylinositol 3‐kinase (PI3K) intracellular signaling pathway in motor neurons, inhibit BDNF‐induced ES. We confirm this finding using a herpes simplex virus (HSV) that expresses the dominant negative p85 subunit of PI3K. Infecting motor neurons with this HSV, but not a control HSV, blocks activation of the PI3K pathway and BDNF‐induced ES. Through the activation of TrkB and the PI3K signaling pathway, BDNF renders developing motor neurons susceptible to glutamate receptor‐mediated cell death.
Angiotensin II‐Induced Decrease in Expression of Inducible Nitric Oxide Synthase in Rat Astroglial Cultures
Authors: Kopnisky Kathy L. , Sumners Colin
Published: March 2003

Abstract: Inducible nitric oxide synthase (iNOS) has been implicated as a mediator of cellular toxicity in a variety of neurodegenerative disorders. Nitric oxide, which is generated in high quantities following induction of iNOS, combines with other oxygen radicals to form highly reactive, death‐inducing compounds. Given the frequency of neuronal death due to neurodegenerative diseases, cerebral trauma, and stroke, it is important to study the mechanisms of regulation of iNOS in the brain. We demonstrated previously that angiotensin II (Ang II) decreases the expression of iNOS produced by bacterial endotoxin or cytokines in cultured astroglia prepared from adult rat brain. Here, we have addressed the mechanisms by which Ang II negatively modulates iNOS. The inhibitory effects of Ang II on lipopolysaccharide‐induced expression of iNOS mRNA and protein and nitrite accumulation were mimicked by the protein kinase C (PKC) activator phorbol 12‐myristate 13‐acetate. Down‐regulation of PKC produced by long‐term treatment of astroglia with phorbol 12‐myristate 13‐acetate abolished the inhibitory effect of Ang II on lipopolysaccharide‐stimulated expression of iNOS mRNA and nitrite accumulation. Finally, the reduction of lipopolysaccharide‐induced nitrite accumulation by Ang II was attenuated by the selective PKC inhibitor chelerythrine. Collectively, these data indicate a role for PKC in the inhibitory actions of Ang II on iNOS expression in cultured astroglia.
Localization of the Heat‐Shock Protein Hsp70 to the Synapse Following Hyperthermic Stress in the Brain
Authors: Bechtold David A. , Rush Sheila J. , Brown Ian R.
Published: March 2003

Abstract: Heat‐shock proteins are induced in response to cellular stress. Although heat‐shock proteins are known to function in repair and protective mechanisms, their relationship to critical neural processes, such as synaptic function, has received little attention. Here we investigate whether the major heat‐shock protein Hsp70 localizes to the synapse following a physiologically relevant increase in temperature in the mammalian nervous system. Our results indicate that hyperthermia‐induced Hsp70 is associated with pre‐ and postsynaptic elements, including the postsynaptic density. The positioning of Hsp70 at the synapse could facilitate the repair of stress‐induced damage to synaptic proteins and also contribute to neuroprotective events at the synapse.
Chronic Stress Induces the Expression of Inducible Nitric Oxide Synthase in Rat Brain Cortex
Authors: Olivenza Raquel , Moro María A. , Lizasoain Ignacio , Lorenzo Pedro , Fernández Ana P. , Rodrigo José , Boscá Lisardo , Leza Juan C.
Published: March 2003

Abstract: Long‐term exposure to stress has detrimental effects on several brain functions in many species, including humans, and leads to neurodegenerative changes. However, the underlying neural mechanisms by which stress causes neurodegeneration are still unknown. We have investigated the role of endogenously released nitric oxide (NO) in this phenomenon and the possible induction of the inducible NO synthase (iNOS) isoform. In adult male rats, stress (immobilization for 6 h during 21 days) increases the activity of a calcium‐independent NO synthase and induces the expression of iNOS in cortical neurons as seen by immunohistochemical and western blot analysis. Three weeks of repeated immobilization increases immunoreactivity for nitrotyrosine, a nitration product of peroxynitrite. Repeated stress causes accumulation of the NO metabolites NO2‐ + NO3‐ (NOx‐) accumulation in cortex, and these changes occur in parallel with lactate dehydrogenase (LDH) release and impairment of glutamate uptake in synaptosomes. Administration of the selective iNOS inhibitor aminoguanidine (400 mg/kg i.p. daily from days 7 to 21 of stress) prevents NOx‐ accumulation in cortex, LDH release, and impairment of glutamate uptake in synaptosomes. Taken together, these findings indicate that a sustained overproduction of NO via iNOS expression may be responsible, at least in part, for some of the neurodegenerative changes caused by stress and support a possible neuroprotective role for specific iNOS inhibitors in this situation.
Identification of an Amino Acid Defining the Distinct Properties of Murine β1 and β3 Subunit‐Containing GABAA Receptors
Authors: Cestari Ismar N. , Min Kyeong T. , Kulli John C. , Yang Jay
Published: March 2003

Abstract: Murine γ‐aminobutyric acid (GABA) type A homomeric receptors made of β1 subunits are profoundly different, when expressed in Xenopus oocytes, from β3 homomeric receptors. Application of the intravenous general anesthetic pentobarbital, etomidate, or propofol to β3 homomeric receptors allows current flow. In contrast, β1 homomers do not respond to any of these agents. Through construction of chimeric β1/β3 receptors, we identified a single amino acid that determines the pharmacological difference between the two β subunits. When the serine residue present in the wild‐type nonresponsive β1 subunit is replaced by an asparagine found in the same position in the β3 subunit, the resulting point‐mutated β1S265N forms receptors responsive to intravenous general anesthetics, like the wild‐type β3 subunits. Conversely, after mutation of the wild‐type β3 to β3N265S, the homomeric receptor loses its ability to respond to these same general anesthetics. Wild‐type‐to‐mutant titration experiments showed that the nonresponsive phenotype is dominant: A single nonresponsive residue within a pentameric receptor is sufficient to render the receptor nonresponsive. In α1βx or α1βxγ2 heteromeric receptors, the same residue manifests as a partial determinant of the degree of potentiation of the GABA‐induced current by some general anesthetics. The location of this amino acid at the extracellular end of the second transmembrane segment, its influence in both homomeric and heteromeric receptor function, and its dominant behavior suggest that this residue of the β subunit is involved in an allosteric modulation of the receptor.
Galanin receptor 1 gene expression is regulated by cyclic AMP through a CREB‐dependent mechanism
Authors: Zachariou Venetia , Georgescu Dan , Kansal Leena , Merriam Priscilla , Picciotto Marina R.
Published: March 2003

The galanin receptor‐1 (GalR1) protein belongs to a family of G protein‐coupled receptors for the neuropeptide galanin (GalR1, GalR2 and GalR3) distributed throughout the central and peripheral nervous system. Activation of galanin receptors by their ligands results in increased feeding, impaired learning, enhanced opiate analgesia and decreased opiate place preference. We have shown that opiate withdrawal, which is known to increase levels of cAMP in the locus coeruleus (LC), results in an increase in the number of galanin binding sites and the level of GalR1 mRNA in the LC. We have isolated a 3.6‐kb fragment 5′ of the inititiation codon of the mouse GalR1 gene and generated a series of deletion mutations of this fragment driving expression of luciferase for use in transient transfection assays in PC12 and Cath.a cell lines. Treatment with forskolin, but not dideoxyforskolin, up‐regulates GalR1 transcription, likely through elevation of cAMP levels. The region between − 1050 and − 700 base pairs upstream of exon one is necessary both for basal activity of the GalR1 promoter and for forskolin‐mediated increases in transcription. The forskolin effect can be blocked by simultaneous mutation of a CRE‐like site and a CRE/DRE‐like site, but not mutation of either site alone. Gel shift and super‐shift experiments demonstrate that the transcription factor CREB can bind to both sites and is likely to be responsible for the cAMP‐mediated increase in GalR1 promoter activity. This study provides a molecular mechanism for the increased GalR1 expression in the LC seen following opiate withdrawal.
Two rat brain Staufen isoforms differentially bind RNA
Authors: Monshausen Michaela , Putz Ulrich , Rehbein Monika , Schweizer Michaela , DesGroseillers Luc , Kuhl, Dietmar , Richter Dietmar , Kindler Stefan
Published: March 2003

In neurones, a limited number of mRNAs is found in dendrites, including transcripts encoding the microtubule‐associated protein 2 (MAP2). Recently, we identified a cis‐acting dendritic targeting element (DTE) in MAP2 mRNAs. Here we used the yeast tri‐hybrid system to identify potential trans‐acting RNA‐binding factors of the DTE. A cDNA clone was isolated that encodes a member of a mammalian protein family that is highly homologous to the Drosophila RNA‐binding protein Staufen. Mammalian Staufen appears to be expressed in most tissues and brain areas. Two distinct rat brain Staufen isoforms, rStau+I6 and rStau‐I6, are encoded by alternatively spliced mRNAs. Both isoforms contain four double‐stranded RNA‐binding domains (dsRBD). In the larger rStau+I6 isoform, six additional amino acids are inserted in the second dsRBD. Although both isoforms interacted with the MAP2‐DTE and various additional RNA fragments in an in vitro north‐western assay, rStau‐I6 exhibited a stronger signal of bound radioactively labelled RNAs as compared with rStau+I6. Using an antibody directed against mammalian Staufen, the protein was detected in somata and dendrites of neurones of the adult rat hippocampus and cerebral cortex. Ultrastructural studies revealed that in dendrites, rat Staufen accumulates along microtubules. Thus in neurones, rat Staufen may serve to link RNAs to the dendritic microtubular cytoskeleton and may thereby regulate their subcellular localization.
Inducible nitric oxide synthase expression in brain cortex after acute restraint stress is regulated by nuclear factor κB‐mediated mechanisms
Authors: Madrigal José L. M. , Moro María A. , Lizasoain Ignacio , Lorenzo Pedro , Castrillo Antonio , Boscá Lisardo , Leza Juan C.
Published: March 2003

The underlying mechanisms by which physical or psychological stress causes neurodegeneration are still unknown. We have demonstrated that the high‐output and long‐lasting synthesizing source of nitric oxide (NO), inducible NO synthase (iNOS), is expressed in brain cortex during stress and that its overexpression accounts for the neurodegenerative changes seen after 3 weeks of repeated stress. Now we have found that acute stress (restraint for 6 h) increases the activity of a calcium‐independent NOS and induces the expression of iNOS in brain cortex in adult male rats. In order to elucidate the possible mechanisms involved in this induction, we studied the role of transcription nuclear factor κB (NF‐κB), which is required for iNOS synthesis. We have observed that an acute restraint stress session stimulates the translocation of the NF‐κB to the nucleus after 4 h and that the administration of the NF‐κB inhibitor pyrrolidine dithiocarbamate [PDTC, 75 and 150 mg/kg intraperitoneally (i.p.)] at the onset of stress inhibits the stress‐induced increase in iNOS expression. Since glutamate release and subsequent NMDA (N‐methyl‐d‐aspartate) receptor activation has been recognized as an early change after exposure to stressful stimuli, and glutamate has been shown to induce iNOS in brain via a NF‐κB‐dependent mechanism, we studied the possible role of excitatory amino acids in the induction of iNOS in our model. Pretreatment with the NMDA receptor antagonist dizocilpine (MK‐801, 0.1 and 0.3 mg/kg i.p.) inhibits the stress‐induced NF‐κB activation as well as the stress‐induced increase in iNOS expression. Taken together, these findings indicate that excitatory amino acids and subsequent activation of NF‐κB account for stress‐induced iNOS expression in cerebral cortex, and support a possible neuroprotective role for specific inhibitors in this situation.
Reaction of oxidized dopamine with endogenous cysteine residues in the human dopamine transporter
Authors: Whitehead Rachel E. , Ferrer Jasmine V. , Javitch Jonathan A. , Justice Joseph B.
Published: March 2003

There is evidence to suggest that dopamine (DA) oxidizes to form dopamine ortho‐quinone (DAQ), which binds covalently to nucleophilic sulfhydryl groups on protein cysteinyl residues. This reaction has been shown to inhibit dopamine uptake, as well as other biological processes. We have identified specific cysteine residues in the human dopamine transporter (hDAT) that are modified by this electron‐deficient substrate analog. DAQ reactivity was inferred from its effects on the binding of [3H]2‐β‐carbomethoxy‐3‐β‐(4‐fluorophenyl)tropane (β‐CFT) to hDAT cysteine mutant constructs. One construct, X5C, had four cysteines mutated to alanine and one to phenylalanine (Cys90A, Cys135A, C306A, C319F and Cys342A). In membrane preparations 1 mm DAQ did not affect [3H]β‐CFT binding to X5C hDAT, in contrast to its effect in wild‐type hDAT in which it reduced the Bmax value by more than half. Wild‐type cysteines were substituted back into X5C, one at a time, and the ability of DAQ to inhibit [3H]β‐CFT binding was assessed. Reactivity of DAQ with Cys90 increased the affinity of [3H]β‐CFT for the transporter, whereas reactivity with Cys135 decreased the affinity of [3H]β‐CFT. DAQ did not change the KD for [3H]β‐CFT binding to wild‐type. The reactivity of DAQ at Cys342 decreased Bmax to the same degree as wild‐type. The latter result suggests that Cys342 is the wild‐type residue most responsible for DAQ‐induced inhibition of [3H]β‐CFT binding.
The cAMP responsive element and CREB partially mediate the response of the tyrosine hydroxylase gene to phorbol ester
Authors: Piech‐Dumas Kristen M. , Best Joseph A. , Chen Yang , Nagamoto‐Combs Kumi , Osterhout Cheryl A. , Tank A. William
Published: March 2003

Tyrosine hydroxylase (TH) gene promoter activity is increased in PC12 cells that are treated with the phorbol ester, 12‐O‐tetradecanoylphorbol 13‐acetate (TPA). Mutagenesis of either the cAMP responsive element (CRE) or the activator protein‐1 element (AP1) within the TH gene proximal promoter leads to a dramatic inhibition of the TPA response. The TH CRE and TH AP1 sites are also independently responsive to TPA in minimal promoter constructs. TPA treatment results in phosphorylation of cAMP responsive element binding protein (CREB) and activation of cAMP‐dependent protein kinase (PKA) in PC12 cells; hence, we tested whether CREB and/or PKA are essential for the TPA response. In CREB‐deficient cells, the response of the full TH gene proximal promoter or the independent response of the TH CRE by itself to TPA is inhibited. The TPA‐inducibility of TH mRNA is also blocked in CREB‐deficient cells. Expression of the PKA inhibitor protein, PKI, also inhibits the independent response of the TH CRE to TPA. Our results support the hypothesis that TPA stimulates the TH gene promoter via signaling pathways that activate either the TH AP1 or TH CRE sites. Both signaling pathways are dependent on CREB and the TH CRE‐mediated pathway is dependent on PKA.
Insulin‐like growth factor‐I is a potential trophic factor for amacrine cells
Authors: Politi Luis E. , Rotstein Nora P. , Salvador Gabriela , Giusto Norma M. , Insua M. Fernanda
Published: March 2003

In this study we show that insulin‐like growth factor (IGF)‐I selectively promotes survival and differentiation of amacrine neurons. In cultures lacking this factor, an initial degeneration pathway, selectively affecting amacrine neurons, led to no lamellipodia development and little axon outgrowth. Cell lysis initially affected 50% of amacrine neurons; those remaining underwent apoptosis leading to the death of approximately 95% of them by day 10. Apoptosis was preceded by a marked increase in c‐Jun expression. Addition of IGF‐I or high concentrations (over 1 µm) of either insulin or IGF‐II to the cultures prevented the degeneration of amacrine neurons, stimulated their neurite outgrowth, increased phospho‐Akt expression and decreased c‐Jun expression. The high insulin and IGF‐II concentrations required to protect amacrine cells suggest that these neurons depend on IGF‐I for their survival, IGF‐II and insulin probably acting through IGF‐I receptors to mimic IGF‐I effects. Inhibition of phosphatidylinositol‐3 kinase (PI 3‐kinase) with wortmannin blocked insulin‐mediated survival. Wortmannin addition had similar effects to IGF‐I deprivation: it prevented neurite outgrowth, increased c‐Jun expression and induced apoptosis. These results suggest that IGF‐I is essential for the survival and differentiation of amacrine neurons, and activation of PI 3‐kinase is involved in the intracellular signaling pathways mediating these effects.