bluuebunny/crossref_metadata_2025 huggingface dataset:
GlutamateNMDA receptor NR1 subunit mRNA expression in Alzheimer's disease
Authors: Hynd Matthew R. , Scott Heather L. , Dodd Peter R.
Published: March 2003

We analyzed the expression profile of two NMDAR1 mRNA isoform subsets, NR10XX and NR11XX, in discrete regions of human cerebral cortex. The subsets are characterized by the absence or presence of a 21‐amino acid N‐terminal cassette. Reverse transcription polymerase chain reaction for NR1 isoforms was performed on total RNA preparations from spared and susceptible regions from 10 pathologically confirmed Alzheimer's disease (AD) cases and 10 matched controls. Primers spanning the splice insert yielded two bands, 342 bp (NR10XX) and 405 bp (NR11XX), on agarose gel electrophoresis. The bands were visualized with ethidium and quantified by densitometry. NR11XX transcript expression was calculated as a proportion of the NR11XX + NR10XX total. Values were significantly lower in AD cases than in controls in mid‐cingulate cortex, p < 0.01, superior temporal cortex, p < 0.01 and hippocampus, p ∼ 0.05. Cortical proportionate NR11XX transcript expression was invariant over the range of ages and areas of controls tested, at ∼50%. This was also true for AD motor and occipital cortex. Proportionate NR11XX expression in AD cingulate and temporal cortex was lower at younger ages and increased with age: this regression was significantly different from that in the homotropic areas of controls. Variations in NR1 N‐terminal cassette expression may underlie the local vulnerability to excitotoxic damage of some areas in the AD brain. Alternatively, changes in NR1 mRNA expression may arise as a consequence of the AD disease process.
Regulation of MARCKS and MARCKS‐related protein expression in BV‐2 microglial cells in response to lipopolysaccharide
Authors: Sunohara Janet R. , Ridgway Neale D. , Cook Harold W. , Byers David M.
Published: March 2003

Myristoylated alanine‐rich C kinase substrate (MARCKS) and MARCKS‐related protein (MRP) have been implicated in membrane‐cytoskeletal events underlying cell adhesion, migration, secretion, and phagocytosis. In BV‐2 microglial cells, lipopolysaccharide (LPS) elicited a dose‐dependent increase in mRNA of both MRP (sixfold) and MARCKS (threefold) with corresponding increases in [3H]myristoylated and immunoreactive protein levels. LPS also produced significant increases in protein kinase C (PKC)‐β twofold and PKC‐ε (1.5‐fold). Pro‐inflammatory cytokines produced by activated microglia (IL‐1β, IL‐6, TNF‐α) did not mimic LPS effects on MARCKS or MRP expression when added individually or in combination. LPS and IFN‐γ produced a synergistic induction of iNOS but not MARCKS or MRP. Induction of MARCKS and MRP by LPS was completely blocked by inhibitors of NF‐κB (PDTC) and protein tyrosine kinases (herbimycin A), partially blocked by the p38 kinase inhibitor SB203580, and unaffected by the MEK inhibitor PD98059. LPS induction of iNOS was considerably more sensitive to all these inhibitors. The Src kinase inhibitor PP2 had no effect, while the closely related inhibitor PP1 actually increased LPS induction of MARCKS and MRP. Our results suggest that MARCKS and MRP may play an important role in LPS‐activated microglia, but are not part of the neuroinflammatory response produced by cytokines.
The caspase‐derived C‐terminal fragment of βAPP induces caspase‐independent toxicity and triggers selective increase of Aβ42 in mammalian cells
Authors: Dumanchin‐Njock Cécile , Da Costa Cristine Alves , Mercken Luc , Pradier Laurent , Checler Frédéric
Published: March 2003

During its physiopathological maturation, the β‐amyloid precursor protein undergoes several distinct proteolytic events by activities called secretases. In Alzheimer's disease, the main histological hallmark called senile plaque is clearly linked to the overproduction of the amyloid peptides Aβ40 and Aβ42, two highly aggregable βAPP‐derived fragments generated by combined cleavages by β‐ and γ‐secretases. Recently, an alternative hydrolytic pathway was described, involving another category of proteolytic activities called caspases, responsible for the production of a 31 amino acids βAPP C‐terminal fragment called C31. C31 was reported to lower the viability of N2a cells but the exact mechanisms mediating C31‐toxicity remained to be established. Here we show that the transient transfection of pSV2 vector encoding C31 lowers by about 80% TSM1 neuronal cells viability. Arguing against a C31‐stimulated apoptotic response, we demonstrate by combined enzymatic and immunological approaches that C31 expression did not modulate basal or staurosporine‐induced caspase 3‐like activity and pro‐caspase‐3 activation. Furthermore, C31 did not modify Bax and p53 expressions, poly‐(ADP‐ribose)‐polymerase cleavage and cytochromectranslocation into the cytosol. However, we established that C31 overexpression triggers selective increase of Aβ42 but not Aβ40 production by HEK293 cells expressing wild‐type βAPP751. Altogether, our data demonstrate that C31 induces a caspase‐independent toxicity in TSM1 neurons and potentiates the pathogenic βAPP maturation pathway by increasing selectively Aβ42 species in wild type‐βAPP‐expressing human cells.
Antidepressant drug treatments induce glial cell line‐derived neurotrophic factor (GDNF) synthesis and release in rat C6 glioblastoma cells
Authors: Hisaoka Kazue , Nishida Akira , Koda Tetsuzo , Miyata Masahiko , Zensho Hidenobu , Morinobu Shigeru , Ohta Mitsuhiro , Yamawaki Shigeto
Published: March 2003

Modulation of neurotrophic factors to protect neurons from damage is proposed as a novel mechanism for the action of antidepressants. However, the effect of antidepressants on modulation of glial cell line‐derived neurotrophic factor (GDNF), which has potent and widespread effects, remains unknown. Here, we demonstrated that long‐term use of antidepressant treatment significantly increased GDNF mRNA expression and GDNF release in time‐ and concentration‐dependent manners in rat C6 glioblastoma cells. Amitriptyline treatment also increased GDNF mRNA expression in rat astrocytes. GDNF release continued for 24 h following withdrawal of amitriptyline. Furthermore, following treatment with antidepressants belonging to several different classes (amitriptyline, clomipramine, mianserin, fluoxetine and paroxetine) significantly increased GDNF release, but which did not occur after treatment with non‐antidepressant psychotropic drugs (haloperidol, diazepam and diphenhydramine). Amitriptyline‐induced GDNF release was inhibited by U0126 (10 µm), a mitogen‐activated protein kinase (MAPK)‐extracellular signal‐related kinase (ERK) kinase (MEK) inhibitor, but was not inhibited by H‐89 (1 µm), a protein kinase A inhibitor, calphostin C (100 nm), a protein kinase C inhibitor and PD 169316 (10 µm), a p38 mitogen‐activated protein kinase inhibitor. These results suggested that amitriptyline‐induced GDNF synthesis and release occurred at the transcriptional level, and may be regulated by MEK/MAPK signalling. The enhanced and prolonged induction of GDNF by antidepressants could promote neuronal survival, and protect neurons from the damaging effects of stress. This may contribute to explain therapeutic action of antidepressants and suggest new strategies of pharmacological intervention.
Study of the brain serotonergic system with labeled α‐methyl‐l‐tryptophan
Authors: Diksic Mirko , Young Simon N.
Published: March 2003

α‐Methyl‐l‐tryptophan (α‐MTrp) is an artificial amino acid and an analog of tryptophan (Trp), the precursor of the neurotransmitter serotonin (5‐HT). In this article we have summarized available data, which suggest that the measurement of the unidirectional uptake of α‐MTrp and its conversion to 5‐HT synthesis rates is a valid approach for the determination of brain 5‐HT synthesis rates. The main feature on which the model is based is the trapping of labeled α‐MTrp in brain tissue. An overview of opposing opinions, which suggest that there is a need for a metabolic conversion of tracer, is also presented and discussed critically. As with all biological modeling there is likely to be room for improvements of the proposed biological model. In addition, there are a limited number of clearly defined circumstances in which the method is confounded by the metabolism of labeled α‐MTrp via the kynurenine pathway. Nonetheless, a significant body of evidence suggests that labeled α‐MTrp is a useful tracer to study brain 5‐HT synthesis in most circumstances. Calculation of 5‐HT synthesis rates depends on the plasma‐free tryptophan concentration, which, according to the balance of arguments in the literature, is a more appropriate parameter than the total‐plasma tryptophan. The method, as proposed by us, can be used in conjunction with autoradiographic measurements in laboratory animals, and with positron emission tomography in large animals and humans. We review studies in animals looking at the normal control of 5‐HT synthesis and the way in which it is altered by drugs, as well as initial studies investigating healthy humans and patients with neuropsychiatric disorders.
Efflux of a suppressive neurotransmitter, GABA, across the blood–brain barrier
Authors: Kakee Atsuyuki , Takanaga Hitomi , Terasaki Tetsuya , Naito Mikihiko , Tsuruo Takashi , Sugiyama Yuichi
Published: March 2003

In this study, GABA efflux transport from brain to blood was estimated by using the brain efflux index (BEI) method. [3H]GABA microinjected into partietal cortex area 2 (Par2) of the rat brain was eliminated from the brain with an apparent elimination half‐life of 16.9 min. The blood–brain barrier (BBB) efflux clearance of [3H]GABA was at least 0.153 mL/min/g brain, which was calculated from the elimination rate constant (7.14 × 10−2 min−1) and the distribution volume in the brain (2.14 mL/g brain). Direct comparison of the apparent BBB influx clearance [3H]GABA (9.29 µL/min/g brain) and the apparent efflux clearance (153 µL/min/g brain) indicated that the efflux clearance was at least 16‐fold greater than the influx clearance. In order to reduce the effect of metabolism in the neuronal cells following intracerebral microinjection, we determined the apparent efflux of [3H]GABA in the presence of nipecotic acid, a GABA transport inhibitor in parenchymal cells, using the BEI method. Under such conditions, the elimination of [3H]GABA across the BBB showed saturation and inhibition by probenecid in the presence of nipecotic acid. Furthermore, the uptake of [3H]GABA by MBEC4 cells was inhibited by GABA, taurine, β‐alanine and nipecotic acid in a concentration‐dependent manner. It is likely that GABA inhibits the first step in the abluminal membrane uptake by brain endothelial cells, and that probenecid selectively inhibits the luminal membrane efflux transport process from the brain capillary endothelial cells based on the in vivo and in vitro evidence. The BBB acts as the efflux pump for GABA to reduce the brain interstitial fluid concentration.
Binding of nerve growth factor to its p75 receptor in stressed cells induces selective IκB‐β degradation and NF‐κB nuclear translocation
Authors: Cosgaya Jose Miguel , Shooter Eric M.
Published: March 2003

Nerve growth factor (NGF) regulates the activity of the transcription factor NF‐κB (nuclear factor‐κB) through its low affinity receptor, p75. In the present study we found that NGF binding to p75 induces nuclear translocation of p65 and increases NF‐κB binding activity in a cell line overexpressing p75, but only after the cells have been subjected to a previous stress. Under physiological conditions, in the absence of stress, NGF is unable to alter p65 nuclear levels. Tumor necrosis factor‐α (TNF‐α) induces a down‐regulation of IκB‐α, ‐β and ‐ε both in physiological and in stress, i.e. serum‐free, conditions. In contrast, NGF only induces the specific degradation of IκB‐β after serum withdrawal, without affecting IκB‐α or ‐ε either in the presence or in the absence of stress. IκB‐β consists of several isoforms, whose relative abundance is regulated by serum withdrawal. NGF does not target all the IκB‐β isoforms with the same potency, being more effective in reducing the levels of the isoforms up‐regulated by serum withdrawal. TRAF‐6 is expressed at the same level under both physiological and stress conditions. These results indicate that NGF is able to induce NF‐κB nuclear translocation by a mechanism that involves specific IκB‐β degradation only after the cells have been subjected to a severe stress.
Effects of selective COX‐1 and ‐2 inhibition on formalin‐evoked nociceptive behaviour and prostaglandin E2 release in the spinal cord
Authors: Tegeder Irmgard , Niederberger Ellen , Vetter Gregor , Bräutigam Lutz , Geisslinger Gerd
Published: March 2003

Nociception evoked prostaglandin (PG) release in the spinal cord considerably contributes to the induction of hyperalgesia and allodynia. To evaluate the relative contribution of cyclooxygenase‐1 (COX‐1) and COX‐2 in this process we assessed the effects of the selective COX‐1 inhibitor SC560 and the selective COX‐2 inhibitor celecoxib on formalin‐evoked nociceptive behaviour and spinal PGE2 release. SC560 (10 and 20 mg/kg) significantly reduced the nociceptive response and completely abolished the formalin‐evoked PGE2 raise. In contrast, celecoxib (10 and 20 mg/kg) was ineffective in both regards, i.e. the flinching behaviour was largely unaltered and the formalin‐induced PGE2 raise as assessed using microdialysis was only slightly, not significantly reduced. This suggests that the formalin‐evoked rapid PG release was primarily caused by COX‐1 and was independent of COX‐2. Mean free spinal cord concentrations of celecoxib during the formalin assay were 32.0 ± 4.5 nm, thus considerably higher than the reported IC50 for COX‐2 (3–7 nm). Therefore, the lack of efficacy of celecoxib is most likely not to be a result of poor tissue distribution. COX‐2 mRNA and protein expression in the spinal cord were not affected by microdialysis alone but the mRNA rapidly increased following formalin injection and reached a maximum at 2 h. COX‐2 protein was unaltered up to 4 h after formalin injection. The time course of COX‐2 up‐regulation suggests that the formalin‐induced nociceptive response precedes COX‐2 protein de novo synthesis and may therefore be unresponsive to COX‐2 inhibition. Considering the results obtained with the formalin model it may be hypothesized that the efficacy of celecoxib in early injury evoked pain may be less than that of unselective NSAIDs.
Two caspase‐2 transcripts are expressed in rat hippocampus after global cerebral ischemia
Authors: Jin Kunlin , Nagayama Tetsuya , Mao Xiaoou , Kawaguchi Kenji , Hickey Robert W. , Greenberg David A. , Simon Roger P. , H. Graham Steven
Published: March 2003

Caspase family genes play a critical role in the initiation and execution of programmed cell death. Programmed cell death is an important contributor to neuronal loss following cerebral ischemia. We have performed a series of experiments to investigate the role of a specific caspase, caspase‐2, in the development of delayed neuronal death following transient global ischemia in the rat. A rat ischemic brain cDNA library was screened, and two splice‐variants of caspase‐2 mRNA were identified, caspase‐2S and caspase‐2L, which were highly homologous with the sequences of human and mouse caspase‐2S and caspase‐2L genes, respectively. RT‐PCR demonstrated an increase in expression of both caspase‐2S and caspase‐2L mRNA at 8, 24 and 72 h of reperfusion after global ischemia. The ratio of the two PCR fragments did not change significantly throughout the time course of reperfusion. Western blot with monoclonal antibody specific to the pro‐apoptotic caspase‐2L splice variant revealed an increase in procaspase‐2 (51 kDa) protein from 4 to 72 h following ischemia compared with sham‐operated controls. Furthermore, an approximately 30‐kDa cleavage product appeared at 8 h and increased with increasing duration of reperfusion. Thus, caspase‐2L is both translated and activated following transient global ischemia. Finally, intraventricular administration of the caspase‐2‐like inhibitor (VDVAD‐FMK) 30 min before induction of ischemia decreased the number of CA1 neurons staining positively for DNA damage (Klenow‐labeling assay) and increased the number of healthy‐appearing CA1 neurons (cresyl violet) compared with vehicle‐treated controls. Taken together, the data suggest that caspase‐2 induction and activation are important mediators of delayed neuronal death following transient global ischemia.
Selective zif268 mRNA induction in the perirhinal cortex of macaque monkeys during formation of visual pair‐association memory
Authors: Tokuyama Wataru , Okuno Hiroyuki , Hashimoto Takanori , Li Yue Xin , Miyashita Yasushi
Published: March 2003

To elucidate the molecular basis of cognitive memory formation in the primate, transcriptional activation during learning of a visual pair‐association (PA) task was evaluated systematically along the occipito‐temporo‐hippocampal pathway in the macaque monkey brain. Split‐brain monkeys were used for intra‐animal comparison, which enables elimination of animal‐to‐animal variation in gene expression. We found that the expression of the mRNA of an immediate‐early gene (IEG), zif268, was up‐regulated selectively in the perirhinal cortex (area 36) during the formation of PA memory compared to that during learning of a visual control task. The mRNA expression levels of another IEG, c‐jun, were not up‐regulated during the PA learning in any cortical areas examined. We also showed that cells strongly expressing zif 268 mRNA accumulated in patches in area 36 during learning of the PA task. As the zif 268 gene encodes a transcription factor, these results suggest that the activation of zif 268 mRNA in area 36 may function as a trigger of the cascade of gene activation that leads to cellular events underlying neuronal reorganization for visual long‐term memory formation.
A non‐amyloidogenic function of BACE‐2 in the secretory pathway
Authors: Fluhrer Regina , Capell Anja , Westmeyer Gil , Willem Michael , Hartung Bianka , Condron Margaret M. , Teplow David B. , Haass Christian , Walter Jochen
Published: March 2003

β‐Site amyloid precursor protein cleavage enzyme (BACE)‐1 and BACE‐2 are members of a novel family of membrane‐bound aspartyl proteases. While BACE‐1 is known to cleave β‐amyloid precursor protein (βAPP) at the β‐secretase site and to be required for the generation of amyloid β‐peptide (Aβ), the role of its homologue BACE‐2 in amyloidogenesis is less clear. We now demonstrate that BACE‐1 and BACE‐2 have distinct specificities in cleavage of βAPP in cultured cells. Radiosequencing of the membrane‐bound C‐terminal cleavage product revealed that BACE‐2 cleaves βAPP in the middle of the Aβ domain between phenylalanines 19 and 20, resulting in increased secretion of APPs‐α‐ and p3‐like products and reduced production of Aβ species. This cleavage can occur in the Golgi and later secretory compartments. We also demonstrate that BACE‐1‐mediated cleavage of βAPP at Asp1 of the Aβ domain can occur as early as in the endoplasmic reticulum, while cleavage at Glu11 occurs in later compartments. These data indicate that the distinct specificities of BACE‐1 and BACE‐2 in their cleavage of βAPP differentially affect the generation of Aβ.
Shp‐2 positively regulates brain‐derived neurotrophic factor‐promoted survival of cultured ventral mesencephalic dopaminergic neurons through a brain immunoglobulin‐like molecule with tyrosine‐based activation motifs/Shp substrate‐1
Authors: Takai Satomi , Yamada Masashi , Araki Toshiyuki , Koshimizu Hisatsugu , Nawa Hiroyuki , Hatanaka Hiroshi
Published: March 2003

To examine the roles of Shp‐2, a cytoplasmic tyrosine phosphatase, in neuronal survival, we generated and used recombinant adenoviruses expressing wild type and phosphatase‐inactive (C/S), phosphatase domain‐deficient (delta P) and constitutively active (D61A and E76A) mutants of Shp‐2. We found that wild‐type Shp‐2 enhanced brain‐derived neurotrophic factor (BDNF)‐promoted survival of cultured ventral mesencephalic dopaminergic neurons. In contrast, the C/S and delta P mutants of Shp‐2 did not affect survival. In addition, the constitutively active D61A and E76A mutants mimicked BDNF and promoted survival. Furthermore, to examine the effects of BIT/SHPS‐1, a substrate of Shp‐2, on the BDNF‐promoted survival, we generated adenovirus vectors expressing wild‐type BIT/SHPS‐1 and its 4F mutant in which all tyrosine residues in the cytoplasmic domain of BIT/SHPS‐1 were replaced with phenylalanine. We found that BDNF‐promoted survival of cultured mesencephalic dopaminergic neurons was enhanced by expression of the 4F mutant but not of wild‐type BIT/SHPS‐1. In addition, we found that co‐expression of wild‐type BIT/SHPS‐1 with Shp‐2 significantly enhanced the survival‐promoting effect of BDNF on cultured mesencephalic dopaminergic neurons. These results indicated that Shp‐2 positively regulates the survival‐promoting effect of BDNF on cultured ventral mesencephalic dopaminergic neurons. Dephosphorylation of BIT/SHPS‐1 by Shp‐2 may participate in BDNF‐stimulated survival signaling.
p‐Methylthioamphetamine and 1‐(m‐chlorophenyl)piperazine, two non‐neurotoxic 5‐HT releasers in vivo, differ from neurotoxic amphetamine derivatives in their mode of action at 5‐HT nerve endings in vitro
Authors: Gobbi M. , Moia M. , Pirona L. , Ceglia I. , Reyes‐Parada M. , Scorza C. , Mennini T.
Published: March 2003

The mechanism underlying the serotoninergic neurotoxicity of some amphetamine derivatives, such as p‐chloroamphetamine (pCA) and 3,4‐methylenedioxymethamphetamine (MDMA), is still debated. Their main acute effect, serotonin (5‐HT) release from nerve endings, involves their interaction with 5‐HT transporters (SERTs), as substrates. Although this interaction is required for the neurotoxic effects, 5‐HT release alone may not be sufficient to induce long‐term 5‐HT deficits. Some non‐neurotoxic compounds, including p‐methylthioamphetamine (MTA) and 1‐(m‐chlorophenyl)piperazine (mCPP), have 5‐HT releasing properties in vivo and in brain slices comparable to that of neurotoxic amphetamine derivatives. We measured 5‐HT release in superfused rat brain synaptosomes preloaded with [3H]5‐HT, a model that distinguishes a releasing effect from reuptake inhibition. MTA and mCPP induced much lower release than pCA and MDMA. The striking difference between our findings in synaptosomes and those obtained in vivo or in brain slices is probably related to a different compartmentalisation of 5‐HT in the different experimental models. Studies in synaptosomes, where the vesicular storage of 5‐HT is predominant, could therefore bring to light differences between neurotoxic and non‐neurotoxic 5‐HT releasing agents which cannot be appreciated in other experimental models and might be useful to identify the mechanisms responsible for the neurotoxicity induced by amphetamine derivatives.
Neuronal‐induced and glutamate‐dependent activation of glial glutamate transporter function
Authors: Poitry‐Yamate Carol L. , Vutskits Laszlo , Rauen Thomas
Published: March 2003

The activity of high‐affinity glutamate transporters is essential for the normal function of the mammalian central nervous system. Using a combined pharmacological, confocal immunocytochemical, enzyme‐based microsensor and fluorescence imaging approach, we examined glutamate uptake and transporter protein localization in single astrocytes of neuron‐containing and neuron‐free microislands prior to pre‐synaptic transmitter secretion and during functional neuronal activity. Here, we report that the presence or absence of neurons strikingly affects the uptake capacity of the astroglial glutamate transporters GLT1 and GLAST1. Induction of transporter function is activated by neurons and this effect is mimicked by pre‐incubation of astrocytes with micromolar concentrations of glutamate. Moreover, increased glutamate transporter activation is reproduced by endogenous release of glutamate via activation of neuronal nicotinic receptors. The increase in transport activity is dependent on neuronal release of glutamate, is associated with the local redistribution (clustering) of GLT1 and GLAST1 but is independent of transporter synthesis and of glutamate receptor activation. Together, these results suggest an activity‐dependent neuronal feedback system for rapid astroglial glutamate transporter regulation where neuron‐derived glutamate is the physiological signal that triggers transporter function.
Trax is a component of the Translin‐containing RNA binding complex
Authors: Finkenstadt Patricia M. , Jeon Mihee , Baraban Jay M.
Published: March 2003

Translin is a nucleic acid binding protein that has been implicated in regulating the targeting and translation of dendritic RNA. In previous studies, we found that Translin and its partner protein, Trax, are components of a gel‐shift complex that is highly enriched in brain extracts. In those studies, we employed a DNA oligonucleotide, GS1, as a probe to label the complex. Translin has also been identified as a component of a gel‐shift complex detected using an RNA oligonucleotide probe, derived from the 3′ UTR of protamine‐2 mRNA. Although we had assumed that these probes labeled the same complex, recent studies indicate that association of Trax with Translin suppresses its RNA binding activity. As these findings challenge this assumption and suggest that the native RNA binding complex does not contain Trax, we have re‐examined this issue. We have found that the gel‐shift complexes labeled with either GS1 or protamine‐2 probes are ‘supershifted’ by addition of Trax antibodies, indicating that both are heteromeric Translin/Trax complexes. In addition, cross‐competition studies provide additional evidence that these probes label the same complex. Furthermore, analysis of recombinant Translin/Trax complexes generated by co‐transfection of Trax with Translin in hEK293T demonstrates that they are labeled with either probe. Although recombinant Translin forms a homomeric nucleic acid binding complex in vitro, our findings indicate that both Trax and Translin are components of the native gel‐shift complex labeled with either GS1 or protamine‐2 probes.
Striatal dopamine–NMDA receptor interactions in the modulation of glutamate release in the substantia nigra pars reticulata in vivo: opposite role for D1 and D2 receptors
Authors: Marti Matteo , Mela Flora , Bianchi Clementina , Beani Lorenzo , Morari Michele
Published: March 2003

Dual probe microdialysis was employed in conscious rats to investigate whether endogenous dopamine is involved in the stimulation of glutamate release in the substantia nigra pars reticulata following striatal NMDA receptor activation. Intrastriatal perfusion with NMDA (1 and 10 µm) facilitated nigral glutamate release (dizocilpine‐ and tetrodotoxin‐sensitive). The D2 dopamine receptor antagonist raclopride increased spontaneous nigral glutamate release and caused a leftward shift in the NMDA sensitivity, lowering NMDA effective concentrations to submicromolar levels. Conversely, the D1 antagonist SCH23390 prevented the effect of NMDA (1 µm) and caused a rightward shift in the NMDA sensitivity. It was tested whether the antagonist effects were due to dopamine receptor blockade or increased tone on D1/D2 receptors. SCH23390 prevented the raclopride‐induced enhancement of spontaneous but not NMDA‐evoked glutamate release while raclopride left unchanged the SCH23390‐induced inhibition. The physiopathological relevance of the dopaminergic modulation was strengthened by perfusing NMDA in the dopamine‐depleted striatum of hemiparkinsonian rats. Nigral glutamate responsiveness to NMDA was enhanced as with raclopride. We conclude that endogenous striatal dopamine regulates both spontaneous and NMDA‐induced nigral glutamate release via an opposite control mediated by D1 facilitatory and D2 inhibitory receptors. Alterations of this control may subserve the motor symptoms of Parkinson's disease.
The neuroprotective agents chlomethiazole and SB203580 inhibit IL‐1β signalling but not its biosynthesis in rat cortical glial cells
Authors: Simi Anastasia , Porsmyr‐Palmertz Margareta , Hjertén Anna , Ingelman‐Sundberg Magnus , Tindberg Niclas
Published: March 2003

Chlomethiazole and pyridinyl imidazole compounds, exemplified by SB203580, are structurally distinct p38 mitogen‐activated protein kinase inhibitors with neuroprotective properties in models of cerebral ischaemia. We have examined their effects in interleukin‐1β (IL‐1β) synthesis, release and signalling in rat cortical glial cells, given the important role of IL‐1β in cerebral ischaemia. We analysed (i) IL‐1β mRNA expression by northern blot, (ii) IL‐1β protein precursor levels within the cells by western blot, and (iii) the levels of the mature IL‐1β protein secreted into the medium by enzyme‐linked immunosorbent assay (ELISA) after treatment of rat cortical glial cells with lipopolysaccharide. While the induction of IL‐1β expression by lipopolysaccharide or by IL‐1β itself was very sensitive to nuclear factor kappa B (NF‐κB) inhibitors, chlomethiazole or SB203580 were nearly without effect, indicating a differential regulation as compared to peripheral cells, e.g. monocytes. In contrast, chlomethiazole and SB203580 potently inhibited the IL‐1β‐induced expression of c‐fos and inducible nitric oxide synthase, as monitored by northern blot and quantitative RT–PCR, respectively. Because IL‐1β‐induced expression of c‐fos and inducible nitric oxide synthase is believed to directly contribute to the pathology of cerebral ischaemic injury, the results suggest a direct mechanism for the neuroprotective effects of chlomethiazole and SB203580, and further establish the anti‐inflammatory properties of chlomethiazole.
Intercommunications between brain capillary endothelial cells and glial cells increase the transcellular permeability of the blood–brain barrier during ischaemia
Authors: Brillault Julien , Berezowski Vincent , Cecchelli Roméo , Dehouck Marie‐Pierre
Published: March 2003

Increased cerebrovascular permeability is an important factor in the development of cerebral oedema after stroke, implicating the blood–brain barrier (BBB). To investigate the effect of hypoxia on the permeability changes, we used a cell culture model of the BBB consisting of a co‐culture of brain capillary endothelial cells and glial cells. When endothelial cells from this co‐culture model were submitted alone to hypoxic conditions, long exposures (48 h) were necessary to result in an increase in endothelial cell monolayer permeability to [3H]inulin. When endothelial cells were incubated in presence of glial cells, a huge increase in permeability occurred after 9 h of hypoxic conditions. Oxygen glucose deprivation (OGD) resulted in a much shorter time (i.e. 2 h) required for an increase in permeability. We have demonstrated that this OGD‐induced permeability increase involves a transcellular rather than a paracellular pathway. Conditioned medium experiments showed that glial cells secrete soluble permeability factors during OGD. However, endothelial cells have to be made sensitive by OGD in order to respond to these glial soluble factors. This work shows that an early cross‐talk between glial and endothelial cells occurs during ischaemic stroke and alters BBB transcellular transport by means of glial factor secretions.
Study of the nematode putative GABA type‐A receptor subunits: evidence for modulation by ivermectin
Authors: Feng Xiao‐Peng , Hayashi Jon , Beech Robin N. , Prichard Roger K.
Published: March 2003

Two alleles of the HG1 gene, which encodes a putative GABA receptor α/γ subunit, were isolated from Haemonchus contortus. These two alleles were shown previously to be associated with ivermectin susceptibility (HG1A) and resistance (HG1E), respectively. Sequence analysis indicates that they differ in four amino acids. To explore the functional properties of the two alleles, a full‐length cDNA encoding the β subunit, a key functional component of the GABA receptor, was isolated from Caenorhabditis elegans (gab‐1, corresponding to the GenBank locus ZC482.1) and coexpressed in Xenopus oocytes with the HG1 alleles. When gab‐1 was coexpressed with either the HG1A allele or the HG1E allele in Xenopus oocytes, γ‐aminobutyric acid (GABA)‐responsive channels with different sensitivity to the agonist were formed. The effects of ivermectin on the hetero‐oligomeric receptors were determined. Application of ivermectin alone had no effect on the receptors. However, when coapplied with 10 µm GABA, ivermectin potentiated the GABA‐evoked current of the GAB‐1/HG1A receptor, but attenuated the GABA response of the GAB‐1/HG1E receptor. We demonstrated that the coexpressed HG1 and GAB‐1 receptors are GABA‐responsive, and provide evidence for the possible involvement of GABA receptors in the mechanism of ivermectin resistance.
Extracellular ATP and nerve growth factor intensify hypoglycemia‐induced cell death in primary neurons: role of P2 and NGFRp75 receptors
Authors: Cavaliere Fabio , Sancesario Giuseppe , Bernardi Giorgio , Volonté Cinzia
Published: March 2003

In this study, we monitored the direct expression of P2 receptors for extracellular ATP in cerebellar granule neurons undergoing metabolism impairment. Glucose deprivation for 30–60 min inhibited P2Y1 receptor protein, only weakly modulated P2X1, P2X2 and P2X3, and up‐regulated by about two‐fold P2X4, P2X7 and P2Y4. The P2X/Y antagonist basilen blue, protecting cerebellar neurons from hypoglycemic cell death, maintained within basal levels only the expression of P2X7 and P2Y4 proteins, but not P2X4 or P2Y1. Glucose starvation transiently increased (up to three‐fold) the expression of NGFRp75 receptor protein and strongly stimulated the extracellular release of nerve growth factor (NGF; about 10‐fold). Exogenously added NGF then augmented hypoglycemic neuronal death by about 60%, increasing the percentage of Höechst‐positive nuclei (from approximately 62 to 95%), reducing lactate dehydrogenase (LDH) release (from about 50 to 14%) and significantly overstimulating the hypoglycemia‐induced expression of P2X7 and P2Y4. Conversely, extracellular ATP augmented hypoglycemic neuronal death by about 80%, reducing the number of Höechst‐positive nuclei (from approximately 62% to 14%), augmenting LDH outflow (by about 30%) and further increasing the hypoglycemia‐induced expression of NGFRp75. Our results indicate that P2 and NGFRp75 receptors are modulated during glucose starvation and that extracellular ATP and NGF drive features of, respectively, necrotic and apoptotic hypoglycemic cell death, aggravating the consequences of metabolism impairment in cerebellar primary neurons.
Nicotine‐induced phosphorylation of Akt through epidermal growth factor receptor and Src in PC12h cells
Authors: Nakayama Hitoshi , Numakawa Tadahiro , Ikeuchi Toshihiko
Published: March 2003

Nicotine treatment triggers calcium influx into neuronal cells, which promotes cell survival in a number of neuronal cells. Phosphoinositide (PI) 3‐kinase and downstream PI3‐kinase target Akt have been reported to be important in the calcium‐mediated promotion of survival in a wide variety of cells. We investigated the mechanisms of nicotine‐induced phosphorylation of Akt in PC12h cells, in comparison with nicotine‐induced ERK phosphorylation. Nicotine induced Akt phosphorylation in a dose‐dependent manner. A nicotinic acetylcholine receptor (nAChR) α7 subunit‐selective inhibitor had no significant effect on nicotine‐induced Akt phosphorylation, while a non‐selective nAChR antagonist inhibited the phosphorylation. L‐type voltage‐sensitive calcium channel (VSCC) antagonists, calmodulin antagonist, and Ca2+/calmudulin‐dependent protein kinase (CaM kinase) inhibitor prevented the nicotine‐induced Akt phosphorylation. Three epidermal growth factor receptor (EGFR) inhibitors prevented the nicotine‐induced phosphorylation of both extracellular signal‐regulated protein kinase (p42/44 MAP kinase, ERK) and Akt. In contrast, an inhibitor of the Src family tyrosine kinase prevented the nicotine‐induced Akt phosphorylation but not ERK phosphorylation. These results suggested that nicotine induces the activation of both PI3‐kinase/Akt and ERK pathways via common pathways including non‐α7‐nAChRs, L‐type VSCC, CaM kinase II and EGFR in PC12h cells, but Src family tyrosine kinases only participate in the pathway to activate Akt.
Characterization and regulation of the 3β‐hydroxysteroid dehydrogenase isomerase enzyme in the rat sciatic nerve
Authors: Coirini H. , Gouézou M. , Delespierre B. , Liere P. , Pianos A. , Eychenne B. , Schumacher M. , Guennoun R.
Published: March 2003

In the peripheral nervous system, progesterone (PROG) has a stimulatory effect on myelination. It could be derived from local synthesis, as Schwann cells in culture express the 3β‐hydroxysteroid dehydrogenase (3β‐HSD) and convert pregnenolone (PREG) to PROG. Although 3β‐HSD mRNA can be detected by RT‐PCR in peripheral nerves, the activity of the enzyme has so far not been demonstrated and characterized in nerve tissue. In this study, we show that homogenates prepared from rat sciatic nerves contain a functional 3β‐HSD enzyme and we have analysed its kinetic properties and its regulation by steroids. The activity of 3β‐HSD in homogenates was evaluated using 3H‐labelled PREG as a substrate and NAD+ as a cofactor, the levels of steroids formed were calculated either by extrapolating the relationship between tritiated peaks obtained by TLC to the initial amount of PREG, or by gas chromatography/mass spectrometry determination. A rapid increase in PROG formation was found between 0 and 50 min of incubation and no further significant changes were observed between 1 and 4 h. The calculated Km value (1.06 ± 0.19 μm) was close to the values described for the3β‐HSD type‐I and type‐IV isoforms. Trilostane, a competitive inhibitor of the 3β‐HSD caused a potent inhibition of the rate of conversion of PREG to PROG (IC50 = 4.06 ± 2.58 μm). When the effects of different steroids were tested, both oestradiol and PROG significantly inhibited the conversion of PREG to PROG.
Vitamin E blocks early events induced by 1‐methyl‐4‐phenylpyridinium (MPP+) in cerebellar granule cells
Authors: González‐Polo Rosa A. , Soler Germán , Alvarez Alberto , Fabregat Isabel , Fuentes José M.
Published: March 2003

Exposure of cerebellar granule cells (CGCs) to 1‐methyl‐4‐phenylpyridinium (MPP+) results in apoptotic cell death, which is markedly attenuated by co‐treatment of CGCs with the radical scavenger vitamin E. Analysis of free radical production and mitochondrial transmembrane potential (ΔΨm), using specific fluorescent probes, showed that MPP+ mediates early radical oxygen species (ROS) production without a loss of ΔΨm. Exposure to MPP+ also produces an early increase in Bad dephosphorylation and translocation of Bax to the mitochondria. These events are accompanied by cytochrome c release from mitochondria to cytosol, which is followed by caspase 3 activation. Exposure of the neurons to vitamin E maintains Bad phosphorylation and attenuates Bax translocation, inhibiting cytochrome c release and caspase activation. MPP+‐mediated cytochrome c release is also prevented by allopurinol, suggesting the participation of xanthine oxidase in the process. Our results indicate that free radicals play an active role in the MPP+‐induced early events that culminate with cell death.
Two pathways for tBID‐induced cytochrome c release from rat brain mitochondria: BAK‐ versus BAX‐dependence
Authors: Brustovetsky Nickolay , Dubinsky Janet M. , Antonsson Bruno , Jemmerson Ronald
Published: March 2003

The mechanisms of truncated BID (tBID)‐induced Cyt c release from non‐synaptosomal brain mitochondria were examined. Addition of tBID to mitochondria induced partial Cyt c release which was inhibited by anti‐BAK antibodies, implicating BAK. Immunoblotting showed the presence of BAK, but not BAX, in brain mitochondria. tBID did not release Cyt c from rat liver mitochondria, which lacked both BAX and BAK. This indicated that tBID did not act independently of BAX and BAK. tBID plus monomeric BAX produced twice as much Cyt c release as did tBID or oligomeric BAX alone. Neither tBID alone nor in combination with BAX induced mitochondrial swelling. In both cases Cyt c release was insensitive to cyclosporin A plus ADP, inhibitors of the mitochondrial permeability transition (mPT). Recombinant Bcl‐xL inhibited Cyt c release induced by tBID alone or in combination with monomeric BAX. Koenig's polyanion, an inhibitor of VDAC, suppressed tBID‐induced Cyt c release from brain mitochondria mediated by BAK but not by BAX. Thus, tBID can induce mPT‐independent Cyt c release from brain mitochondria by interacting with exogenous BAX and/or with endogenous BAK that may involve VDAC. In contrast, neither adenylate kinase nor Smac/DIABLO was released from isolated rat brain mitochondria via BAK or BAX.
Polyamine transport, accumulation, and release in brain
Authors: Masuko Takashi , Kusama‐Eguchi Kuniko , Sakata Kaori , Kusama Tadashi , Chaki Shigeyuki , Okuyama Shigeru , Williams Keith , Kashiwagi Keiko , Igarashi Kazuei
Published: March 2003

Cycling of polyamines (spermine and spermidine) in the brain was examined by measuring polyamine transport in synaptic vesicles, synaptosomes and glial cells, and the release of spermine from hippocampal slices. It was found that membrane potential‐dependent polyamine transport systems exist in synaptosomes and glial cells, and a proton gradient‐dependent polyamine transport system exists in synaptic vesicles. The glial cell transporter had high affinities for both spermine and spermidine, whereas the transporters in synaptosomes and synaptic vesicles had a much higher affinity for spermine than for spermidine. Polyamine transport by synaptosomes was inhibited by putrescine, agmatine, histidine, and histamine. Transport by glial cells was also inhibited by these four compounds and additionally by norepinephrine. On the other hand, polyamine transport by synaptic vesicles was inhibited only by putrescine and histamine. These results suggest that the polyamine transporters present in glial cells, neurons, and synaptic vesicles each have different properties and are, presumably, different molecular entities. Spermine was found to be accumulated in synaptic vesicles and was released from rat hippocampal slices by depolarization using a high concentration of KCl. Polyamines, in particular spermine, may function as neuromodulators in the brain.
Pharmacological and immunohistochemical characterization of the APJ receptor and its endogenous ligand apelin
Authors: Medhurst Andrew D. , Jennings Carol A. , Robbins Melanie J. , Davis Robert P. , Ellis Catherine , Winborn Kim Y. , Lawrie Kenneth W. M. , Hervieu Guillaume , Riley Graham , Bolaky Jane E. , Herrity Nicole C. , Murdock Paul , Darker John G.
Published: March 2003

Apelin peptides have recently been identified to be the endogenous ligands for the G protein‐coupled receptor APJ. However, little is known about the physiological roles of this ligand‐receptor pairing. In the present study we investigated the pharmacology of several apelin analogues at the human recombinant APJ receptor using radioligand binding and functional assays. This has led to the identification of key residues in the apelin peptide required for functional potency and binding affinity through structure–activity studies. In particular, we have identified that replacement of leucine in position 5, or arginine in position 2 and 4 of the C‐terminal apelin peptide, apelin‐13, resulted in significant changes in pharmacology. We also investigated the detailed localization of pre‐proapelin and APJ receptor mRNA in a wide range of human, rat and mouse tissues using quantitative RT–PCR, and carried out a detailed immunohistochemical study of the distribution of the APJ receptor in rat brain and spinal cord. Interestingly, the APJ receptor was not only co‐localized in white matter with GFAP in the spinal cord, but was also clearly localized on neurones in the brain, suggesting that this receptor and its peptide may be involved in a wide range of biological process yet to be determined.
Methyl‐β‐cyclodextrin but not retinoic acid reduces EAAT3‐mediated glutamate uptake and increases GTRAP3‐18 expression
Authors: Butchbach Matthew E. R. , Guo Hong , Lin Chien‐liang Glenn
Published: March 2003

The Na+‐dependent glutamate transporter EAAT3 facilitates glutamate uptake into neurons as well as many other cell types. GTRAP3‐18 (JWA, Arl6ip5) is a novel protein that interacts with EAAT3 and negatively modulates EAAT3‐mediated glutamate uptake. Previous studies suggest that retinoic acid (RA) decreases Na+‐dependent glutamate uptake and increases GTRAP3‐18 protein expression. However, the RA used in those studies was complexed with methyl‐β‐cyclodextrin (MeβCD). In the present study we found that MeβCD, but not RA, significantly reduced Na+‐dependent EAAT3‐mediated [3H]glutamate uptake in human embryonic kidney 293 (HEK293) cells. MeβCD also significantly increased GTRAP3‐18 protein expression in HEK293 cells as well as in rat hypothalamic neuron cultures. Intracerebroventricular administration of MeβCD to the mouse brain resulted in a significant increase in GTRAP3‐18 immunoreactivity in the hippocampus and cerebral cortex. In conclusion, we have shown that MeβCD reduces EAAT3‐mediated glutamate uptake and induces the expression of GTRAP3‐18 protein.
Acidosis has opposite effects on neuronal survival during hypoxia and reoxygenation
Authors: Almaas Runar , Pytte Morten , Lindstad Julie K. , Wright Marianne , Saugstad Ola Didrik , Pleasure David , Rootwelt Terje
Published: March 2003

To study the effect of extracellular acidosis on apoptosis and necrosis during ischemia and reoxygenation, we exposed human post‐mitotic NT2‐N neurones to oxygen and glucose deprivation (OGD) followed by reoxygenation. In some experiments, pH of the cell medium was lowered to 5.9 during either OGD or reoxygenation or both. Staurosporine, used as a positive control for apoptosis, caused Poly(ADP‐ribose)‐polymerase (PARP) cleavage and nuclear fragmentation, but no PARP cleavage and little fragmentation were seen after OGD. Low molecular weight DNA fragments were found after staurosporine treatment, but not after OGD. No protective effect of caspase inhibitors was seen after 3 h of OGD and 21 h of reoxygenation, but after 45 h of reoxygenation caspase inhibition induced a modest improvement in 3‐(4,5‐dimethylthiazol‐2‐yl)2,5‐diphenyltetrazolium bromide (MTT) cleavage. While acidosis during OGD accompanied by neutral medium during reoxygenation protected the neurones (MTT: 228 ± 117% of neutral medium,p < 0.001), acidosis during reoxygenation only was detrimental (MTT: 38 ± 25%,p < 0.01). We conclude that apoptotic mechanisms play a minor role after OGD in NT2‐N neurones. The effect of acidosis on neuronal survival depends on the timing of acidosis, as acidosis was protective during OGD and detrimental during reoxygenation.