bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis and Characterization of Binding of 5‐[76Br] Bromo‐3‐[[2(S)‐Azetidinyl]methoxy]pyridine, a Novel Nicotinic Acetylcholine Receptor Ligand, in Rat Brain
Authors: Sihver Wiebke , Fasth Karl‐Johan , Horti Andrew G. , Koren Andrei O. , Bergström Mats , Lu Li , Hagberg Gisela , Lundqvist Hans , Dannals Robert F. , London Edythe D. , Nordberg Agneta , Långström Bengt
Published: March 2003

Abstract : 5‐[76Br]Bromo‐3‐[[2(S)‐azetidinyl]methoxy]‐pyridine ([76Br]BAP), a novel nicotinic acetylcholine receptor ligand, was synthesized using [76Br]bromide in an oxidative bromodestannylation of the corresponding trimethylstannyl compound. The radiochemical yield was 25%, and the specific radioactivity was on the order of 1 Ci/μmol. The binding properties of [76Br]BAP were characterized in vitro and in vivo in rat brain, and positron emission tomography (PET) experiments were performed in two rhesus monkeys. In association experiments on membranes of the cortex and thalamus, >90% of maximal specific [76Br]BAP binding was obtained after 60 min. The dissociation half‐life of [76Br]BAP was 51 ± 6 min in cortical membranes and 56 ± 3 min in thalamic membranes. Saturation experiments with [76Br]BAP revealed one population of binding sites with dissociation constant (KD) values of 36 ± 9 and 30 ± 9 pM in membranes of cortex and thalamus, respectively. The maximal binding site density (Bmax) values were 90 ± 17 and 207 ± 33 fmol/mg in membranes of cortex and thalamus, respectively. Scatchard plots were nonlinear, and the Hill coefficients were <1, suggesting the presence of a lower‐affinity binding site. In vitro autoradiography studies showed that binding of [76Br]BAP was high in the thalamus and presubiculum, moderate in the cortex and striatum, and low in the cerebellum and hippocampus. A similar pattern of [76Br]BAP accumulation was observed by ex vivo autoradiography. In vivo, binding of [76Br]BAP in whole rat brain was blocked by preinjection of (S)(‐)‐nicotine (0.3 mg/kg) by 27, 52, 68, and 91% at survival times of 10, 25, 40, 120, and 300 min, respectively. In a preliminary PET study in rhesus monkeys, the highest [76Br]BAP uptake was found in the thalamus, and radioactivity was displaceable by ~60% with cytisine and by 50% with (S)(‐)‐nicotine. The data of this study indicate that [76Br]BAP is a promising radioligand for the characterization of nicotinic acetylcholine receptors in vivo.
Malonate‐Induced Generation of Reactive Oxygen Species in Rat Strium Depends on Dopamine Release but Not on NMDA Receptor Activation
Authors: Ferger Boris , Eberhardt Olaf , Teismann Peter , De Groote Carmen , Schulz Jörg B.
Published: March 2003

Abstract : Intrastriatal injection of the reversible succinate dehydrogenase inhibitor malonate produces both energy depletion and striatal lesions similar to that seen in cerebral ischemia and Huntington’s disease. The mechanisms of neuronal cell death involve secondary excitotoxicity and the generation of reactive oxygen species. Here, we investigated the effects of dopamine on malonate‐induced generation of hydroxyl radicals and striatal lesion volumes. Using in vivo microdialysis, we found that malonate induced a 94‐fold increases in extracellular striatal dopamine concentrations. This was paralleled by an increase in the generation of hydroxyl radicals. Prior unilateral lesioning of the nigrostriatal dopaminergic pathway by focal injection of 6‐hydroxydopamine blocked the malonate‐induced increase in dopamine concentrations and the generation of hydroxyl radicals and attenuated the lesion volume. In contrast, the NMDA receptor antagonist MK‐801 attenuated malonate‐induced lesion volumes but did not block the generation of hydroxyl radicals. Thus, the dopaminergic and glutamatergic pathways are essential in the pathogenesis of malonate‐induced striatal lesions. Our results suggest that the malonate‐induced release of dopamine but not NMDA receptor activation mediates hydroxyl radical formation.
Mouse Postganglionic Sympathetic Neurons
Authors: Trendelenburg Anne‐Ulrike , Gaiser Eugen Gerhard , Cox Sandra Leagh , Meyer Angelika , Starke Klaus
Published: March 2003

Abstract : Basic properties of noradrenaline release were studied in

primary cultures of thoracolumbar postganglionic sympathetic neurons taken

from 1‐3‐day‐old NMRI mice. After 7 days in vitro, the cultures were

preincubated with [3H]noradrenaline and then superfused and

stimulated electrically. Conventional trains of pulses (for example, 36 pulses

at 3 Hz) as well as single pulses and brief high‐frequency trains (for

example, four pulses at 100 Hz) elicited a well‐measurable overflow of

tritium, which was abolished by 0.3 μM tetrodotoxin or omission of

Ca2+, but not changed by 1 μM rauwolscine. In trains of

one, two, four, six, eight, or 10 pulses at 3 Hz, the evoked overflow of

tritium remained constant from pulse to pulse at 1.3 mM

Ca2+, but declined slightly at 2.5 mM Ca2+.

Tetraethylammonium at 10 mM selectively increased the overflow

elicited by small pulse numbers and especially by a single pulse. In trains of

10 pulses delivered at 0.3, 1, 3, 10, 30, or 100 Hz, the evoked overflow of

tritium increased from 0.3 to 30 Hz and then declined at 100 Hz. This

relationship was particularly pronounced at low Ca2+ concentrations

(for example, 0.3 mM) Tetraethylammonium at 10 mM

selectively increased the overflow elicited by low frequencies of stimulation.

It is concluded that primary cultures of mouse postganglionic sympathetic

neurons can be used to investigate release of [3H]noradrenaline. The release is well measurable, even upon a single electrical pulse. It agrees with release in intact sympathetically innervated tissues in a number of fundamental properties, including the pulse number and frequency dependence. The preparation may be of special interest in conjunction with genetic manipulations in the donor animals.
Amphetamine‐Induced Glutamate Efflux in the Rat Ventral Tegmental Area Is Prevented by MK‐801, SCH 23390, and Ibotenic Acid Lesions of the Prefrontal Cortex
Authors: Wolf Marina E. , Xue Chang‐Jiang
Published: March 2003

Abstract : We showed previously that amphetamine challenge produces a delayed increase in glutamate efflux in the ventral tegmental area of both naive and chronic amphetamine‐treated rats. The present study examined the mechanisms underlying this response. The NMDA receptor antagonist MK‐801 (0.1 mg/kg, i.p.) or the D1 dopamine receptor antagonist SCH 23390 (0.1 mg/kg, i.p.), given 30 min before acute amphetamine (5 mg/kg, i.p.), prevented amphetamine‐induced glutamate efflux. Neither antagonist by itself altered glutamate efflux. Ibotenic acid lesions of the prefrontal cortex similarly prevented amphetamine‐induced glutamate efflux, while producing a trend toward decreased basal glutamate levels (82.8% of sham group). Previous work has shown that the doses of NMDA and D1 receptor antagonists used in this study prevent the induction of behavioral sensitization when coadministered repeatedly with amphetamine, and that identical prefrontal cortex lesions performed before repeated amphetamine prevent the induction of ambulatory sensitization. Thus, treatments that prevent acute amphetamine from elevating glutamate efflux in the ventral tegmental area also prevent repeated amphetamine from eliciting behavioral sensitization. These findings suggest that repeated elevation of glutamate levels during a chronic amphetamine regimen may contribute to the cascade of neuroadaptations within the ventral tegmental area that enables the induction of sensitization.
Molecular Cloning of a Cell Cycle Regulation Gene Cyclin H from Ischemic Rat Brain
Authors: Jin Kunlin , Nagayama Tetsuya , Chen Jun , Stetler Anne R. , Kawaguchi Kenji , Simon Roger P. , Graham Steven H.
Published: March 2003

Abstract : Gene expression plays an important role in determining the fate of neurons after ischemia. To identify additional genes that promote survival or execute programmed cell death in ischemic neurons, a subtractive cDNA library was constructed from hippocampus of rats subjected to global ischemia. With use of a differential screening technique, a cDNA was identified that was upregulated after ischemia. The cDNA was found to have high homology with human cyclin H at both the nucleotide level (89%) and the amino acid level (93%). Northern blotting detected cyclin H mRNA in nonischemic and ischemic brains. In situ hybridization studies revealed that cyclin H message was found in hippocampal neurons in nonischemic brain. After ischemia, expression was increased primarily in the dentate gyrus and CA3 regions of hippocampus. Expression of cyclin H protein, detected by western blotting of hippocampal tissue, was increased after global ischemia, but expression of cyclins B1 and D1 and other related cell cycle genes (Cdk7 and Cdc2) was not increased. Cyclin H immunoreactivity was found exclusively within neurons. After ischemia, there was increased immunoreactivity within neurons in dentate gyrus, CA3, and cortex. Thus, cyclin H is expressed in normal postmitotic neurons and expression is increased in neurons that are ischemic yet survive. These results suggest that cyclin H may have functions in neurons other than cell cycle regulation, including other known functions such as DNA repair.
Activity‐Dependent Neurotrophic Factor Peptide (ADNF9) Protects Neurons Against Oxidative Stress‐Induced Death
Authors: Glazner Gordon W. , Boland Andre , Dresse Albert E. , Brenneman Douglas E. , Gozes Illana , Mattson Mark P.
Published: March 2003

Abstract : Activity‐dependent neurotrophic factor (ADNF) and a

14‐amino acid fragment of this peptide (sequence VLGGGSALLRSIPA) protect

neurons from death associated with an array of toxic conditions, including

amyloid β‐peptide, N‐methyl‐d‐aspartate,

tetrodotoxin, and the neurotoxic HIV envelope coat protein gp120. We report

that an even smaller, nine‐amino acid fragment (ADNF9) with the sequence

SALLRSIPA potently protects cultured embryonic day 18 rat hippocampal neurons

from oxidative injury and neuronal apoptosis induced by FeSO4 and trophic factor withdrawal. Among the characteristics of this protection are maintenance of mitochondrial function and a reduction in accumulation of intracellular reactive oxygen species.
Chronic Exposure to Ammonia Alters Pathways Modulating Phosphorylation of Microtubule‐Associated Protein 2 in Cerebellar Neurons in Culture
Authors: Sáez Rosana , Llansola Marta , Felipo Vicente
Published: March 2003

Abstract : Hyperammonemia is considered the main cause for the

neurological alterations found in hepatic failure. However, the mechanisms by

which high ammonia levels impair cerebral function are not well understood. It

has been shown that chronic hyperammonemia impairs signal transduction

pathways associated with NMDA receptors and also alters phosphorylation of

some neuronal proteins. The aim of the present work was to analyze the effects

of chronic exposure to ammonia on phosphorylation of microtubule‐associated

protein 2 (MAP‐2) in intact neurons in culture and to assess whether

modulation of MAP‐2 phosphorylation by glutamate receptor‐associated

transduction pathways is altered in neurons chronically exposed to ammonia. It

is shown that chronic exposure to ammonia increases basal phosphorylation of

MAP‐2 by ~70%. This effect seems to be due to a decreased tonic activation of

NMDA receptors and of calcineurin. Chronic exposure to ammonia also alters the

modulation of MAP‐2 phosphorylation by NMDA receptors and metabotropic

glutamate receptors. In neurons exposed to ammonia, treatment with NMDA for 30

min induced a significant decrease in phosphorylation of MAP‐2. Activation of

metabotropic glutamate receptors with

(1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid significantly increased phosphorylation of MAP‐2 in control neurons, whereas in neurons exposed to ammonia the response was the opposite, with 1‐aminocyclopentane‐1,3‐dicarboxylic acid inducing a dephosphorylation of MAP‐2. These results indicate that ammonia alters significantly signal transduction pathways associated with different types of glutamate receptors. This would lead therefore to significant alterations in glutamatergic neurotransmission, which would contribute to the neurological alterations found in hyperammonemia and in hepatic encephalopathy.
Neuronal Apoptosis in Mouse Trisomy 16
Authors: Bambrick Linda L. , Krueger Bruce K.
Published: March 2003

Abstract: Hippocampal neurons from the trisomy 16 (Ts16) mouse, a potential animal model of Down’s syndrome (trisomy 21) and neurodegenerative disorders such as Alzheimer’s disease (AD), die at an accelerated rate in vitro. Here, we present evidence that the accelerated neuronal death in Ts16 occurs by apoptosis, as has been reported for neurons in AD. First, the nuclei of dying Ts16 neurons are pyknotic and undergo DNA fragmentation, as revealed by terminal transferase‐mediated dUTP nick end‐labeling. Second, the accelerated death of Ts16 neurons is prevented by inhibitors of the caspase family of proteases, which are thought to act at a late, obligatory step in the apoptosis pathway. In the presence of maximally effective concentrations of caspase inhibitors, Ts16 neuron survival was indistinguishable from that of control neurons. These results suggest that overexpression of one or more genes on mouse chromosome 16 leads to caspase‐mediated apoptosis in Ts16 neurons.
Human Bcl‐2 Protects Against AMPA Receptor‐Mediated Apoptosis
Authors: Cheung Nam S. , Beart Philip M. , Pascoe Catherine J. , John Christopher A. , Bernard Ora
Published: March 2003

Abstract: Dysfunctions of the
(S)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)
subtype of ionotropic receptor for the brain's major excitatory
neurotransmitter, L‐glutamate, occur in various neurological conditions. We
have previously demonstrated that AMPA receptor‐mediated excitotoxicity occurs
by apoptosis and here examined the influence of the expression of cell death
repressor gene Bcl‐2 on this excitotoxic insult. Using neuronal
cortical cultures prepared from transgenic mice expressing the human
Bcl‐2 gene, the influence of Bcl‐2 on AMPA receptor‐mediated
neuronal death was compared with that seen with staurosporine and
H2O2. At day 6 cultures were exposed to AMPA (0.1‐100
μM), and cellular injury was analyzed 48 h after insult using
phase‐contrast microscopy, a
3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide viability assay,
and DNA staining with 4,6‐diamidino‐2‐phenylindole and Sytox Green. AMPA
produced a concentration‐dependent increase in cell death that was
significantly attenuated by human Bcl‐2. AMPA (3 μM)
increased the number of apoptotic nuclei to 60% of control in wild‐type
cultures, and human Bcl‐2 significantly decreased the number of
apoptotic nuclei to 30% of AMPA‐treated cultures. Human Bcl‐2 only
provided significant neuroprotection against neuronal injury induced by low
concentrations of staurosporine (1‐10 nM) and
H2O2 (0.1‐30 μM) and where neuronal death
was by apoptosis, but not against H2O2‐induced necrosis.
Our findings indicate that overexpression of Bcl‐2 in primary cultured neurons protects in an insult‐dependent manner against AMPA receptor‐mediated apoptosis, whereas protection was not seen against more traumatic insults. This study provides new insights into the molecular therapeutics of neurodegenerative conditions.
Enhancement of Anandamide Formation in the Limbic Forebrain and Reduction of Endocannabinoid Contents in the Striatum of Δ9‐Tetrahydrocannabinol‐Tolerant Rats
Authors: Di Marzo V. , Berrendero F. , Bisogno T. , González S. , Cavaliere P. , Romero J. , Cebeira M. , Ramos J. A. , Fernández‐Ruiz J. J.
Published: March 2003

Abstract: Recent studies have shown that the pharmacological
tolerance observed after prolonged exposure to synthetic or plant‐derived
cannabinoids in adult rats is accompanied by down‐regulation/desensitization
of brain cannabinoid receptors. However, no evidence exists on possible
changes in the contents of the endogenous ligands of cannabinoid receptors in
the brain of cannabinoid‐tolerant rats. The present study was designed to
elucidate this possibility by measuring, by means of isotope dilution gas
chromatography/mass spectrometry, the contents of both anandamide
(arachidonoylethanolamide; AEA) and its biosynthetic precursor,
N‐arachidonoylphosphatidylethanolamine (NArPE), and
2‐arachidonoylglycerol (2‐AG) in several brain regions of adult male rats
treated daily with Δ9‐tetrahydrocannabinol
(Δ9‐THC) for a period of 8 days. The areas analyzed included
cerebellum, striatum, limbic forebrain, hippocampus, cerebral cortex, and
brainstem. The same regions were also analyzed for cannabinoid receptor
binding and WIN‐55,212‐2‐stimulated
guanylyl‐5′‐O‐(γ‐[35S]thio)‐triphosphate
([35S]GTPγS) binding to test the development of the well
known down‐regulation/desensitization phenomenon. Results were as follows: As
expected, cannabinoid receptor binding and WIN‐55,212‐2‐stimulated
[35S]GTPγS binding decreased in most of the brain areas of
Δ9‐THC‐tolerant rats. The only region exhibiting no changes
in both parameters was the limbic forebrain. This same region exhibited a
marked (almost fourfold) increase in the content of AEA after 8 days of
Δ9‐THC treatment. By contrast, the striatum exhibited a
decrease in AEA contents, whereas no changes were found in the brainstem,
hippocampus, cerebellum, or cerebral cortex. The increase in AEA contents
observed in the limbic forebrain was accompanied by a tendency of NArPE levels
to decrease, whereas in the striatum, no significant change in NArPE contents
was found. The contents of 2‐AG were unchanged in brain regions from
Δ9‐THC‐tolerant rats, except for the striatum where they dropped significantly. In summary, the present results show that prolonged activation of cannabinoid receptors leads to decreased endocannabinoid contents and signaling in the striatum and to increased AEA formation in the limbic forebrain. The pathophysiological implications of these findings are discussed in view of the proposed roles of endocannabinoids in the control of motor behavior and emotional states.
Role for Dopamine in Malonate‐Induced Damage In Vivo in Striatum and In Vitro in Mesencephalic Cultures
Authors: Moy L. Y. , Zeevalk G. D. , Sonsalla P. K.
Published: March 2003

Abstract:Defects in mitochondrial energy metabolism have been implicated in the pathology of several neurodegenerative disorders. In addition, the reactive metabolites generated from the metabolism and oxidation of the neurotransmitter dopamine (DA) are thought to contribute to the damage to neurons of the basal ganglia. We have previously demonstrated that infusions of the metabolic inhibitor malonate into the striata of mice or rats produce degeneration of DA nerve terminals. In the present studies, we demonstrate that an intrastriatal infusion of malonate induces a substantial increase in DA efflux in awake, behaving mice as measured by in vivo microdialysis. Furthermore, pretreatment of mice with tetrabenazine (TBZ) or the TBZ analogue Ro 4‐1284 (Ro‐4), compounds that reversibly inhibit the vesicular storage of DA, attenuates the malonate‐induced DA efflux as well as the damage to DA nerve terminals. Consistent with these findings, the damage to both DA and GABA neurons in mesencephalic cultures by malonate exposure was attenuated by pretreatment with TBZ or Ro‐4. Treatment with these compounds did not affect the formation of free radicals or the inhibition of oxidative phosphorylation resulting from malonate exposure alone. Our data suggest that DA plays an important role in the neurotoxicity produced by malonate. These findings provide direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular DA levels and indicate that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through a mechanism involving DA.
Differential Assembly of Human Tau Isoforms in the Presence of Arachidonic Acid
Authors: King Michelle E. , Gamblin T. Chris , Kuret Jeff , Binder Lester I.
Published: March 2003

Abstract: Six tau isoforms arise from the alternative splicing of a single gene in humans. Insoluble, filamentous deposits of tau protein occur in a number of neuro‐degenerative diseases, and in some of these diseases, the deposition of polymers enriched in certain tau isoforms has been documented. Because of these findings, we have undertaken studies on the efficacy of fatty acid‐induced polymerization of the individual tau isoforms found in the adult human CNS. The polymerization of each tau isoform in the presence of two concentrations of arachidonic acid indicated that isoforms lacking N‐terminal exons e2 and e3 formed small, globular oligomers that did not go on to elongate into straight (SF) or paired helical (PHF) filaments under our buffer conditions. The polymerization of all isoforms containing e2 or e2 and e3 occurred readily at a high arachidonic acid concentration. Conversely, at a lower arachidonic acid concentration, only tau isoforms containing four microtubule binding repeats assembled well. Under all buffer conditions employed, filaments formed from three of the isoforms containing e2 and e3 resembled SFs in morphology but began to form PHF‐like structures following extended incubation at 37°C. These results indicate that polymerization of the intact tau molecule may be facilitated by e2 and e3. Moreover, tau isoforms containing three versus four microtubule binding repeats display different assembly properties depending on the solvent conditions employed.
Hydrophobic Protein that Copurifies with Human Brain Acetylcholinesterase
Authors: Navaratnam Dhasakumar S. , Fernando F. Shama , Priddle John D. , Giles Kurt , Clegg Sheila M. , Pappin Darryl J. , Craig Ian , Smith A. David
Published: March 2003

Abstract: The mechanism of attachment of acetylcholinesterase (AChE) to neuronal membranes in interneuronal synapses is poorly understood. We have isolated, sequenced, and cloned a hydrophobic protein that copurifies with AChE from human caudate nucleus and that we propose forms a part of a complex of membrane proteins attached to this enzyme. It is a short protein of 136 amino acids and has a molecular mass of 18 kDa. The sequence contains stretches of both hydrophobic and hydrophilic amino acids and two cysteine residues. Analysis of the genomic sequence reveals that the coding region is divided among five short exons. Fluorescence in situ hybridization localizes the gene to chromosome 6p21.32‐p21.2. Northern blot analysis shows that this gene is widely expressed in the brain with an expression pattern that parallels that of AChE.
Deficiency of Inducible Nitric Oxide Synthase Protects Against MPTP Toxicity In Vivo
Authors: Dehmer Thomas , Lindenau Jörg , Haid Sibylle , Dichgans Johannes , Schulz Jörg B.
Published: March 2003

Abstract: MPTP produces clinical, biochemical, and neuropathologic changes reminiscent of those that occur in idiopathic Parkinson's disease (PD). In the present study we show that MPTP treatment led to activation of microglia in the substantia nigra pars compacta (SNpc), which was associated and colocalized with an increase in inducible nitric oxide synthase (iNOS) expression. In iNOS‐deficient mice the increase of iNOS expression but not the activation of microglia was blocked. Dopaminergic SNpc neurons of iNOS‐deficient mice were almost completely protected from MPTP toxicity in a chronic paradigm of MPTP toxicity. Because the MPTP‐induced decrease in striatal concentrations of dopamine and its metabolites did not differ between iNOS‐deficient mice and their wild‐type littermates, this protection was not associated with a preservation of nigrostriatal terminals. Our results suggest that iNOS‐derived nitric oxide produced in microglia plays an important role in the death of dopaminergic neurons but that other mechanisms contribute to the loss of dopaminergic terminals in MPTP neurotoxicity. We conclude that inhibition of iNOS may be a promising target for the treatment of PD.
Retinoic Acid‐Mediated Enhancement of the Cholinergic/Neuronal Nitric Oxide Synthase Phenotype of the Medial Septal SN56 Clone
Authors: Personett David , Fass Uwe , Panickar Kiran , McKinney Michael
Published: March 2003

Abstract: It is unclear what mechanisms lead to the degeneration of basal forebrain cholinergic neurons in Alzheimer’s or other human brain diseases. Some brain cholinergic neurons express neuronal nitric oxide (NO) synthase (nNOS), which produces a free radical that has been implicated in some forms of neurodegeneration. We investigated nNOS expression and NO toxicity in SN56 cells, a clonal cholinergic model derived from the medial septum of the mouse basal forebrain. We show here that, in addition to expressing choline acetyltransferase (ChAT), SN56 cells express nNOS. Treatment of SN56 cells with retinoic acid (RA; 1 μM) for 48 h increased ChAT mRNA (+126%), protein (+88%), and activity (+215%) and increased nNOS mRNA (+98%), protein (+400%), and activity (+15%). After RA treatment, SN56 cells became vulnerable to NO excess generated with S‐nitro‐N‐acetyl‐DL‐penicillamine (SNAP) and exhibited increased nuclear DNA fragmentation that was blocked with a caspase‐3 inhibitor. Treatment with dexamethasone, which largely blocked the RA‐mediated increase in nNOS expression, or inhibition of nNOS activity with methylthiocitrulline strongly potentiated the apoptotic response to SNAP in RA‐treated SN56 cells. Caspase‐3 activity was reduced when SNAP was incubated with cells or cell lysates, suggesting that NO can directly inhibit the protease. Thus, whereas RA treatment converts SN56 cells to a proapoptotic state sensitive to NO excess, endogenously produced NO appears to be anti‐apoptotic, possibly by tonically inhibiting caspase‐3.
Molecular Cloning of a Novel Brain‐Type Na+‐Dependent Inorganic Phosphate Cotransporter
Authors: Aihara Yasuo , Mashima Hirosato , Onda Hideaki , Hisano Setsuji , Kasuya Hidetoshi , Hori Tomokatsu , Yamada Shirou , Tomura Hideaki , Yamada Yuichiro , Inoue Ituro , Kojima Itaru , Takeda Jun
Published: March 2003

Abstract: We have isolated a human cDNA encoding a protein, designated DNPI, that shows 82% amino acid identity and 92% similarity to the human brain‐specific Na+‐dependent inorganic phosphate (Na+/Pi) cotransporter (BNPI), which is localized exclusively to neuron‐rich regions. Expression of DNPI mRNA in Xenopus oocytes resulted in a significant increase in Na+‐dependent Pi transport, indicating that DNPI is a novel Na+/Pi cotransporter. Northern blot analysis shows that DNPI mRNA is expressed predominantly in brain, where the highest levels are observed in medulla, substantia nigra, subthalamic nucleus, and thalamus, all of which express BNPI mRNA at low levels. In contrast, DNPI mRNA is expressed at low levels in cerebellum and hippocampus, where BNPI mRNA is expressed at high levels. No hybridizing signal for DNPI mRNA is observed in the glia‐rich region of corpus callosum. In other regions examined, both mRNAs are moderately or highly expressed. These results indicate that BNPI and DNPI, which coordinate Na+‐dependent Pi transport in the neuron‐rich regions of the brain, may form a new class within the Na+/Pi cotransporter family.
Ubiquitination and Degradation of the Zebrafish Paired‐Like Homeobox Protein Vsx‐1
Authors: Kurtzman Aaron L. , Gregori Luisa , Haas Arthur L. , Schechter Nisson
Published: March 2003

Abstract : Vsx‐1 is a paired‐like : CVC homeobox protein

dynamically expressed during zebrafish development. Previous results indicate

that Vsx‐1 influences bipolar cell differentiation and maintenance of these

cells in the adult retina. To understand the developmental regulation of this

transcription factor, we investigated ubiquitination as a possible

posttranslational mechanism. In vitro, Vsx‐1 was conjugated with multiple

ubiquitin moieties. Proteasome inhibitors and added ubiquitin increased the

accumulation of Vsx‐1‐ubiquitinn complexes and stabilized unmodified Vsx‐1. Also, in transiently transfected COS‐7 cells, Vsx‐1 is ubiquitinated, and pulse‐chase experiments show that Vsx‐1 proteolysis occurs. Vsx‐1 proteins with C‐terminal deletions retained the capacity for initial modification by ubiquitin but lost the capacity for efficient chain elongation. These results show that Vsx‐1 is a substrate of the ubiquitin/proteasome pathway and suggest that C‐terminal sequences of Vsx‐1 are critical for ubiquitin chain elongation. In addition, our findings suggest that ubiquitin‐dependent proteolysis regulates Vsx‐1 during zebrafish retinal development.
Isolation and Characterization of Novel Presenilin Binding Protein
Authors: Kashiwa Atsushi , Yoshida Hirotaka , Lee Soon , Paladino Toni , Liu Yuanbin , Chen Qi , Dargusch Richard , Schubert David , Kimura Hideo
Published: March 2003

Approximately 50% of familial Alzheimer's disease (AD) cases are linked to the presenilin (PS) gene. This suggests that an altered function of mutated PSs accounts for a fundamental process leading to AD. Here we identify a new PS binding protein, PBP, which is highly expressed in cerebral cortex and hippocampus. immunohistochemical studies and cell fractionation analysis show that PBP redistributes from cytoplasm to membranes in the presence of PS. In addition, PBP is deficient in the soluble fraction of sporadic AD brains.
Modulation of the Phosphorylation and Activity of Calcium/Calmodulin‐Dependent Protein Kinase II by Zinc
Authors: Lengyel Imre , Fieuw‐Makaroff Sabine , Hall Amanda L , Sim Alistair T R , Rostas John A P , Dunkley Peter R
Published: March 2003

Calcium/calmodulin‐dependent protein kinase II (CaMPK‐II) is a key regulatory enzyme in living cells. Modulation of its activity, therefore, could have a major impact on many cellular processes. We found that Zn2+ has multiple functional effects on CaMPK‐II. Zn2+

generated a Ca2+/CaM‐independent activity that correlated with the

autophosphorylation of Thr286, inhibited Ca2+/CaM

binding that correlated with the autophosphorylation of Thr306, and

inhibited CaMPK‐II activity at high concentrations that correlated with the

autophosphorylation of Ser279. The relative level of

autophosphorylation of these three sites was dependent on the concentration of

zinc used. The autophosphorylation of at least these three sites, together

with Zn2+ binding, generated an increased mobility form of CaMPK‐II

on sodium dodecyl sulfate gels. Overall, autophosphorylation induced by

Zn2+ converts CaMPK‐II into a different form than the binding of

Ca2+/CaM. In certain nerve terminals, where Zn2+ has

been shown to play a neuromodulatory role and is present in high

concentrations, Zn2+ may turn CaMPK‐II into a form that would be unable to respond to calcium signals.
The Triakontatetraneuropeptide (TTN) Stimulates Thymidine Incorporation in Rat Astrocytes Through Peripheral‐Type Benzodiazepine Receptors
Authors: Gandolfo Pierrick , Patte Christine , Leprince Jérôme , Régo Jean‐Luc Do , Mensah‐Nyagan Ayikoe Guy , Vaudry Hubert , Tonon Marie‐Christine
Published: March 2003

Astrocytes and astrocytoma cells actively express the diazepam‐binding inhibitor (DBI) gene, suggesting that DBI‐processing products may regulate glial cell activity. In the present study, we have investigated the possible effect of one of the DBI‐derived peptides, the triakontatetraneuropeptide (TTN), on [3H]thymidine incorporation in cultured rat astrocytes.

Reversed‐phase HPLC analysis of incubation media indicated that TTN is the

major form of DBI‐derived peptides released by cultured astrocytes. At very

low concentrations (10‐14‐10‐11M), TTN induced

a dose‐dependent increase in [3H]thymidine incorporation, whereas

at higher concentrations (10‐10‐10‐5M) the

effect of TTN gradually declined. In the same range of concentrations, the

specific peripheral‐type benzodiazepine receptor (PBR) agonist Ro 5‐4864

mimicked the bell‐shaped stimulatory effect of TTN on [3H]thymidine

incorporation. The PBR antagonist PK11195 (10‐6M)

suppressed the stimulatory action of both TTN and Ro 5‐4864 on

[3H]thymidine incorporation, whereas the central‐type

benzodiazepine receptor antagonist flumazenil (10‐6M) had no effect. The present study demonstrates that the endozepine TTN stimulates DNA synthesis in rat glial cells through activation of PBRs. These data strongly suggest that TTN exerts an autocrine/paracrine stimulatory effect on glial cell proliferation.
Chronic Morphine Treatment and Withdrawal Increase Extracellular Levels of Norepinephrine in the Rat Bed Nucleus of the Stria Terminalis
Authors: Fuentealba José Antonio , Forray María Inés , Gysling Katia
Published: March 2003

Extracellular levels of norepinephrine (NE) and glutamate (Glu) in the ventral bed nucleus of the stria terminalis (vBNST) of saline‐ and chronic morphine‐treated rats, with or without withdrawal, were studied by means of the in vivo microdialysis technique in anesthetized rats. In addition, the tissue concentration of NE was studied at different rostrocaudal levels of the vBNST. Chronic morphine treatment significantly increased extracellular levels of NE, but not Glu, in vBNST. At 48 h after naloxone‐induced morphine withdrawal there was a further significant increase in the extracellular levels of NE, but not Glu, in vBNST. The presence of UK 14304, an α2‐adrenergic agonist, induced a significant decrease in NE extracellular levels in all experimental groups. In contrast, UK 14304 induced a significant decrease in Glu extracellular levels only in saline‐treated rats. The results also show that the vBNST presents a rostrocaudal gradient of NE and contains 9.4% of total brain NE. The increase in NE extracellular levels in vBNST induced by chronic morphine treatment and the further increase in NE levels 48 h after naloxone‐induced morphine withdrawal suggest that NE in vBNST may be involved in the pharmacological effects of chronic morphine and withdrawal.
Modulation of Hypothalamic NMDA Receptor Function by Cyclic AMP‐Dependent Protein Kinase and Phosphatases
Authors: Nijholt Ingrid , Blank Thomas , Liu Allen , Kügler Heidemarie , Spiess Joachim
Published: March 2003

In the present study we investigated the modulation of hypothalamic NMDA receptor‐mediated currents by cyclic AMP‐dependent protein kinase (PKA) using the two‐electrode voltage‐clamp technique in Xenopus oocytes injected

with rat hypothalamic mRNA. Application of forskolin, which activates PKA by

means of cyclic AMP stimulation, caused a transient increase of NMDA‐induced

currents, whereas the inactive forskolin analogue 1,9‐dideoxyforskolin had no

effect. Incubation of oocytes with a membrane‐permeable analogue of cyclic

AMP, 8‐bromoadenosine 3′,5′ ‐cyclic monophosphate, potentiated

NMDA responses even more prominently than with forskolin. NMDA‐induced

currents recorded from Xenopus oocytes injected with cRNA encoding the NMDA receptor subunits NR1, NR2A, and/or NR2B, mainly found in rat hypothalamus, were not affected by PKA activation but were increased by protein kinase C (PKC) stimulation. It is interesting that inhibition of endogenous protein phosphatase 1 and/or 2A by calyculin A resulted in a similar enhancement of hypothalamic NMDA‐induced currents. Preinjection of oocytes with calyculin A impeded the PKA‐ but not the PKC‐mediated potentiation of hypothalamic NMDA‐induced currents. We propose the involvement of an additional third messenger in the PKA effect, which acts most likely via the inhibition of tonically active protein phosphatase 1 and/or 2A.
Effects of Nitrobenzylthioinosine on Neuronal Injury, Adenosine Levels, and Adenosine Receptor Activity in Rat Forebrain Ischemia
Authors: Parkinson F E , Zhang Y W , Shepel P N , Greenway S C , Peeling J , Geiger J D
Published: March 2003

Adenosine levels increase in brain during cerebral ischemia, and adenosine has receptor‐mediated neuroprotective effects. This study was performed to test the hypothesis that nitrobenzylthioinosine (NBMPR), a selective and potent inhibitor of one adenosine transporter subtype termed ENT1, or es, can protect against ischemic neuronal injury by enhancing

adenosine levels and potentiating adenosine receptor‐mediated effects,

including attenuation of the cellular production and release of tumor necrosis

factor‐α (TNF‐α). In rats, the phosphorylated prodrug form of

NBMPR, NBMPR‐phosphate, or saline was administered by intracerebroventricular

injection 30 min before forebrain ischemia. Seven days following the ischemic

episode, rats were killed, and neuronal damage in the CA1 region of the

hippocampus was assessed. The number of pyramidal neurons was significantly

(p < 0.001) greater in the NBMPR‐P treatment group. A trend toward

protection was still evident at 28 days postreperfusion. Adenosine increased

significantly during ischemia to levels eight‐ to 85‐fold above basal. NBMPR‐P

treatment did not cause statistically significant increases in ischemic

adenosine levels ; however, this treatment tended to increase adenosine levels

in all brain regions at 7 min postreperfusion. Ischemia‐induced expression of

TNF‐α was not altered by NBMPR‐P treatment, and the nonselective

adenosine receptor antagonist 8‐(p‐sulfophenyl) theophylline did not abolish the neuroprotective effects of NBMPR‐P treatment. These data indicate that NBMPR can protect CA1 pyramidal neurons from ischemic death without statistically significant effects on adenosine levels or adenosine receptor‐mediated inhibition of the proinflammatory cytokine TNF‐α.
Regulation of Neurotransmitter Release by Metabotropic Glutamate Receptors
Authors: Cartmell Jayne , Schoepp Darryle D.
Published: March 2003

Abstract: The G protein‐coupled metabotropic glutamate (mGlu)

receptors are differentially localized at various synapses throughout the

brain. Depending on the receptor subtype, they appear to be localized at

presynaptic and/or postsynaptic sites, including glial as well as neuronal

elements. The heterogeneous distribution of these receptors on glutamate and

nonglutamate neurons/cells thus allows modulation of synaptic transmission by

a number of different mechanisms. Electrophysiological studies have

demonstrated that the activation of mGlu receptors can modulate the activity

of Ca2+ or K+ channels, or interfere with release

processes downstream of Ca2+ entry, and consequently regulate neuronal synaptic activity. Such changes evoked by mGlu receptors can ultimately regulate transmitter release at both glutamatergic and nonglutamatergic synapses. Increasing neurochemical evidence has emerged, obtained from in vitro and in vivo studies, showing modulation of the release of a variety of transmitters by mGlu receptors. This review addresses the neurochemical evidence for mGlu receptor‐mediated regulation of neurotransmitters, such as excitatory and inhibitory amino acids, monoamines, and neuropeptides.
Amphetamine Increases Glutamate Efflux in the Rat Ventral Tegmental Area by a Mechanism Involving Glutamate Transporters and Reactive Oxygen Species
Authors: Wolf Marina E. , Xue Chang‐Jiang , Li Yong , Wavak David
Published: March 2003

Abstract: We have shown that amphetamine produces a delayed and
sustained increase in glutamate levels in the ventral tegmental area, a region
containing dopamine cell bodies important in acute and chronic effects of
amphetamine administration. The present study characterized the mechanism
underlying amphetamine‐induced glutamate efflux. It was abolished by the
glutamate uptake inhibitor dihydrokainate, but unaffected by perfusion with a
low Ca2+/high Mg2+ solution, implicating glutamate
transporters. Because reactive oxygen species inhibit glutamate uptake, we
examined the effect of amphetamine on hydroxyl radical formation by perfusing
with D‐phenylalanine (5 mM) and monitoring p‐tyrosine
production. Although no increase in hydroxyl radical formation was detected,
D‐phenylalanine completely prevented the amphetamine‐induced increase in
glutamate efflux, as did systemic injection of another trapping agent,
α‐phenyl‐N‐tert‐butyl nitrone (60 mg/kg). Thus,
amphetamine‐induced glutamate efflux may involve reactive oxygen species. In
other studies, we found that repeated coadministration of
α‐phenyl‐N‐tert‐butyl nitrone with amphetamine attenuated the development of behavioral sensitization. This supports prior results indicating that the increase in glutamate efflux produced by each amphetamine injection in a chronic regimen is important in triggering drug‐induced adaptations in ventral tegmental area dopamine neurons, and that such adaptations may in part represent a response to metabolic and oxidative stress
The Single‐Stranded DNA‐ and RNA‐Binding Proteins Pur α and Pur β Link BC1 RNA to Microtubules Through Binding to the Dendrite‐Targeting RNA Motifs
Authors: Ohashi Sachiyo , Kobayashi Shunsuke , Omori Akira , Ohara Susumu , Omae Akiko , Muramatsu Takahiro , Li Yanmei , Anzai Kaijiro
Published: March 2003

Abstract: Neural BC1 RNA is distributed in neuronal dendrites as RNA—protein complexes (BC1 RNPs) containing Translin. In this study, we demonstrated that the single‐stranded DNA‐ and RNA‐binding protein pur α and its isoform, pur β, which have been implicated in control of DNA replication and transcription, linked BC1 RNA to microtubules (MTs). The binding site was within the 5′ proximal region of BC1 RNA containing putative dendrite‐targeting RNA motifs rich in G and U residues, suggesting that in the cytoplasm of neurons, these nuclear factors are involved in the BC1 RNA transport along dendritic MTs. The pur proteins were not components of BC1 RNP but appeared to associate with MTs in brain cells. Therefore, it is suggested that they may transiently interact with the RNP during transport. In this respect, the interaction of pur proteins with BC1 RNA could be regulated by the Translin present within the RNP, because the binding mode of these two classes of proteins (pur proteins and Translin) to the dendrite‐targeting RNA motifs was mutually exclusive. As the motifs are well conserved in microtubule‐associated protein 2a/b mRNA as well, the pur proteins may also play a role(s) in the dendritic transport of a subset of mRNAs.
Evidence for a G Protein‐Coupled γ‐Hydroxybutyric Acid Receptor
Authors: Snead O. Carter
Published: March 2003

Abstract:γ‐Hydroxybutyric acid (GHB) is a naturally occurring
metabolite of GABA that has been postulated to exert ubiquitous
neuropharmacological effects through GABAB receptor
(GABABR)‐mediated mechanisms. The alternative hypothesis that GHB
acts via a GHB‐specific, G protein‐coupled presynaptic receptor that is
different from the GABABR was tested. The effect of GHB on regional
and subcellular brain adenylyl cyclase in adult and developing rats was
determined and compared with that of the GABABR agonist
(‐)‐baclofen. Also, using guanosine
5′‐O‐(3‐[35S]thiotriphosphate)
([35S]GTPγS) binding and low‐Km GTPase
activity as markers the effects of GHB and (‐)‐baclofen on G protein activity
in the brain were determined. Neither GHB nor baclofen had an effect on basal
cyclic AMP (cAMP) levels. GHB significantly decreased forskolin‐stimulated
cAMP levels by 40‐50% in cortex and hippocampus but not thalamus or
cerebellum, whereas (‐)‐baclofen had an effect throughout the brain. The
effect of GHB on adenylyl cyclase was observed in presynaptic and not
postsynaptic subcellular tissue preparations, but the effect of baclofen was
observed in both subcellular preparations. The GHB‐induced alteration in
forskolin‐induced cAMP formation was blocked by a specific GHB antagonist but
not a specific GABABR antagonist. The (‐)‐baclofen‐induced
alteration in forskolin‐induced cAMP formation was blocked by a specific
GABABR antagonist but not a specific GHB antagonist. The negative
coupling of GHB to adenylyl cyclase appeared at postnatal day 21, a
developmental time point that is concordant with the developmental appearance
of [3H]GHB binding in cerebral cortex, but the effects of
(‐)‐baclofen were present by postnatal day 14. GHB and baclofen both
stimulated [35S]GTPγS binding and low‐Km
GTPase activity by 40‐50%. The GHB‐induced effect was blocked by GHB
antagonists but not by GABABR antagonists and was seen only in
cortex and hippocampus. The (‐)‐baclofen‐induced effect was blocked by
GABABR antagonists but not by GHB antagonists and was observed
throughout the brain. These data support the hypothesis that GHB induces a G
protein‐mediated decrease in adenylyl cyclase via a GHB‐specific G
protein‐coupled presynaptic receptor that is different from the
GABABR.
Structural Characteristics of Human Substantia Nigra Neuromelanin and Synthetic Dopamine Melanins
Authors: Double K. L. , Zecca L. , Costi P. , Mauer M. , Griesinger C. , Ito S. , Ben‐Shachar D. , Bringmann G. , Fariello R. G. , Riederer P. , Gerlach M.
Published: March 2003

Abstract: Neuromelanin (NM) is a complex polymer pigment foundprimarily in the dopaminergic neurons of the human substantia nigra. Thestructure of NM is only partially characterized, and its synthesis pathwayremains unknown. We used nuclear magnetic and infrared spectroscopy to examinethe structure of human NM isolated from the substantia nigra compared withsynthetic dopamine melanins. Biochemical analyses were used to investigateproteinaceous and dopaminergic components in these samples. Following acidhydrolysis of NM samples, small amounts of DOPA, dopamine, and a variety ofamino acids were measured. These findings suggest a peptide component in NMstructure. NM also appears to contain a variety of unidentified structuralcomponents possibly derived from the oxidation of dopamine. Human NM differsstructurally from synthetic dopamine melanin, but both human and synthetic NMinclude an aromatic backbone. It is interesting that both human NM andsynthetic melanin also contain a large proportion of aliphatic structures. Ourresults suggest that NM is a more complex pigment than synthetic dopaminemelanin formed via dopamine autoxidation alone.
Induction of Matrix Metalloproteinase MMP‐9 (92‐kDa Gelatinase) by Retinoic Acid in Human Neuroblastoma SKNBE Cells
Authors: Chambaut‐Guérin Anne‐Marie , Hérigault Sabine , Rouet‐Benzineb Patricia , Rouher Caroline , Lafuma Chantal
Published: March 2003

Abstract: Retinoic acid (RA) has been shown to induce human neuroblastoma SKNBE cell differentiation into a neuronal phenotype. Whether this neuronal differentiation is associated with modulation of matrix gelatinase [matrix metalloproteinase (MMP)‐2 and MMP‐9] expression was investigated in SKNBE cell cultures exposed to RA for 14 days. Their differentiation into a neuronal phenotype was typified by neural cell adhesion molecule and growth‐associated protein‐43 expression. Gelatinase expression was assessed by gel zymography, quantitative RT‐PCR, and immunocytochemistry. Neuronal markers were located in neurites and ganglion‐like clusters of neuronal cells induced upon RA exposure. MMP‐2 expression was constitutive and remained unchanged at both the mRNA and protein levels in response to RA, tumor necrosis factor‐α (TNFα), or phorbol 12‐myristate 13‐acetate (PMA) treatment. In contrast, MMP‐9 was inducible by RA, TNFα, or PMA. MMP‐9 was progressively enhanced by RA as a function of time exposure until day 14. The addition of TNFα or PMA potentiated RA‐induced MMP‐9 expression with a synergic maximal effect at day 14 of RA exposure. Immunoreactive MMP‐9 was located early in outgrowing neurites, but only at day 14 of RA exposure in extensive neuritic networks. Taken together, the correlation between the MMP‐9 expression by SKNBE cells and the time scale of their differentiation into a neuronal phenotype allowed us to propose that MMP‐9 could participate in the neurite growth process and cell migration and organization into ganglion‐like clusters.